Development of a multiplex PCR assay for the detection of metallo-beta-lactamase genes in Pseudomonas aeruginosa / Desarrollo de un PCR múltiple para la detección de genes metalo-beta-lactamasa en Pseudomonas aeruginosa

SUMMARY: The appearance of Pseudomonas aeruginosa strains with multi-resistance to antibiotics is a clinical problem of great relevance. The methods for detecting these resistances are laborious and slow, which is a complication when treating patients promptly. In this work, we developed a simple method for simultaneous detection of several carbapenem resistance genes using a multiplex PCR assay. The PCR assay developed, followed by electrophoretic separation of fragments, allows to simultaneously identify the presence of 6 antibiotic resistance genes: bla-VIM (261 bp), bla-IMP (587 bp), bla-SPM (648 bp), bla-GIM-1 (753 bp), bla-NDM-1 (813 bp) and bla-KPC (882 bp). We analyzed 7 clinical isolates of P. aeruginosa obtained in Chile, finding the resistance genes bla-VIM, bla-IMP, bla-SPM, bla-GIM, and bla-NDM in 5 of them. We found a perfect correlation between the detection of various resistance genes by PCR and the results obtained by antibiograms. Interestingly, 2 of the strains possessed 3 different resistance genes simultaneously. Finally, in this work, we found the presence of 3 genes never described before in clinical isolates of P. aeruginosa in Chile (bla-IMP, bla-SPM, and bla-GIM-1). We developed a rapid multiplex PCR test for the simultaneous detection of up to 6 antibiotic resistance genes of the metallo-β-lactamase family in P. aeruginosa.

La aparición de cepas de Pseudomonas aeruginosa con resistencias a diversos antibióticos es un problema clínico de gran relevancia. Los métodos de detección de dichas resistencias son laboriosos y lentos, lo que genera una complicación al momento de tratar a los pacientes oportunamente. En este trabajo desarrollamos un método simple de detección simultánea de varios genes de resistencia a carbapenem, mediante un sistema de PCR múltiple. El ensayo de PCR desarrollado, seguido de una separación electroforética de los amplicones, permite distinguir simultáneamente la presencia de 6 genes de resistencia a antibióticos: bla-VIM (261 pb), bla-IMP (587 pb), bla-SPM (648 pb), bla-GIM-1 (753 pb), bla-NDM-1 (813 pb) y bla-KPC (882 pb). Analizamos 7 aislados clínicos obtenidos en Chile, encontrando en 5 de ellos los genes de resistencia bla-VIM, bla-IMP, bla-SPM, bla-GIM y bla-NDM. Encontramos una perfecta correlación entre la detección de diversos genes de resistencia y los resultados obtenidos mediante antibiogramas. Interesantemente, 2 de las cepas mostraron poseer simultáneamente 3 genes de resistencia distintos. Por último, en este trabajo encontramos la presencia de 3 genes nunca antes descritos en aislados clínicos de P. aeruginosa en Chile (bla-IMP, bla-SPM y bla-GIM-1). Hemos desarrollado un test rápido de PCR múltiple, para la detección simultánea de hasta 6 genes de resistencia a antibióticos de la familia.a de las metallo-b-lactamases en P. aeruginosa.

Effect of calcium on Pseudomonas aeruginasa and Bacillus cereus metabolites / Efeito do cálcio nos metabólitos de Pseudomonas aeruginasa e Bacillus cereus

The effects of Calcium (Ca+²) on virulence and some parameters should be analyzed in this study. Pseudomonas aeruginosa Gram (-) and Bacillus cereus Gram (+) were used. Both bacteria are soil bacteria. In this study; the effect of Ca+² on protease, amylase, LasB elastolytic assay, H2O2, pyorubin and biofilm on metabolites of these bacteria were investigated during 24 hour time. In this study, the effect of Ca+² on the production of some secondary metabolites on P. aeruginosa and B. cereus was investigated and presented for the first time by us.

Os efeitos do cálcio (Ca+²) na virulência e alguns parâmetros devem ser analisados neste estudo. Pseudomonas aeruginosa Gram (-) e Bacillus cereus Gram (+) foram usados. Ambas as bactérias são bactérias do solo. Neste estudo, o efeito do Ca+² sobre a protease, amilase, ensaio elastolítico LasB, H2O2, piorubina e biofilme nos metabólitos dessas bactérias foram investigados durante 24 horas. Neste estudo, o efeito do Ca+² na produção de alguns metabólitos secundários em P. aeruginosa e B. cereus foi investigado e apresentado pela primeira vez por nós.

Obtención de hidrolizados proteicos de leguminosas usando una proteasa recombinante de Pseudomonas aeruginosa M211 / Obtaining protein hydrolysates from leguminous using a recombinant protease from Pseudomonas aeruginosa M211

El género Pseudomonas es una fuente importante de proteasas; sin embargo, su uso está restringido en la industria alimentaria. El clonaje permite aprovechar la capacidad catalítica de estas enzimas mediante su producción en microorganismos inocuos. Por otro lado, las leguminosas son fuentes ricas en proteínas, a partir de las cuales se pueden obtener compuestos con valor agregado mediante procesos de hidrólisis enzimática. En este estudio, se produjo y caracterizó una proteasa recombinante (PT4) alcalina y termoestable de Pseudomonas aeruginosa M211, para la obtención de hidrolizados proteicos de leguminosas. Para ello, el gen de la proteasa se clonó en el vector pJET1.2/blunt utilizando E. coli DHalfa como hospedero. El análisis de la secuencia nucleotídica parcial de la proteasa indicó un 99 % de similitud con Peptidasas de la Familia M4 de Pseudomonas aeruginosa. La enzima recombinante presentó un peso molecular de 80 kDa, demostró ser activa y estable en condiciones alcalinas y termófilas con un pH y temperatura óptimos de 8 y 60 °C, respectivamente, y fue inhibida por EDTA. Además, hidrolizó proteínas de semillas de Glycine max, Phaseolus lunatus, Lupinus mutabilis y Erythrina edulis, obteniéndose fracciones peptídicas menores a 40 kDa. Esta proteasa recombinante se podría utilizar en la elaboración de hidrolizados proteicos funcionales a partir proteínas de distintas fuentes y residuos agroalimentarios.

The genus Pseudomonas is an important source of proteases; however, in the food industry the use of this bacterium is restricted. Cloning allows for the use of the proteolytic activity of Pseudomonas proteases through their production in innocuous microorganisms. Leguminous are protein-rich sources from which value-added compounds can be obtained through enzymatic hydrolysis. In this study, an alkaline and thermostable recombinant protease (PT4) from Pseudomonas aeruginosa M211 was cloned and characterized in order to obtain protein hydrolysates from leguminous. Therefore, protease gene was cloned into the pJET1.2 / blunt vector using E. coli DHalpha as a host. Analysis of protease partial nucleotide sequence showed 99% homology with Peptidases M4 Family from Pseudomonas aeruginosa. The molecular weight of the recombinant enzyme was 80 kDa, it was active and stable under alkaline and thermophilic conditions, presented an optimum pH and temperature of 8 and 60 °C, respectively, and was inhibited by EDTA. In addition, it hydrolysed Glycine max, Phaseolus lunatus, Lupinus mutabilis y Erythrina edulis proteins, obtaining peptide fractions less than 40 kDa. This recombinant protease could be used in the elaboration of functional hydrolysates using protein from different sources and agricultural waste.

Caracterización de metalo-β-lactamasas en aislados clínicos de Pseudomonas aeruginosa recuperados de pacientes hospitalizados en el Hospital Militar Central / Characterization of metallo-β-lactamase in clinical isolates of Pseudomonas aeruginosa retrieved from patients hospitalized in the Central Military Hospital

Con el objetivo de caracterizar y determinar la frecuencia de genes que codifican metalo-β-lactamasas (MBL) en aislados clínicos de Pseudomonas aeruginosa (P. aeruginosa), se realizó un estudio transversal en el Hospital Militar Central (HMC) de Lima, Perú, entre enero y setiembre del 2016. Se analizaron 76 aislados de P. aeruginosa con sensibilidad disminuida a carbapenémicos y resistentes a ceftazidima. La detección fenotípica de MBL se realizó por el método de sinergia de doble disco entre imipenem y meropenem frente al ácido etilendiaminotetraacético (EDTA), y la identificación de los genes por reacción en cadena de la polimerasa. De los 76 aislados, 24 (31,6 %) fueron positivos para MBL por el método fenotípico, confirmándose genéticamente todos. Se encontró el gen blaIMP en 23/24 (95,8 %) y blaVIM en 1/24 (4,2 %). Ningún aislado presentó blaNDM. El gen blaIMP resultó ser el más frecuente entre los aislados clínicos de P. aeruginosa no sensibles a carbapenémicos en el HMC.

hat codify metallo-β-lactamases (MBL) in clinical isolates of Pseudomona aeruginosa (P. aeruginosa), a cross-sectional study was conducted in the Central Military Hospital (HMC) of Lima, Peru, between January and September, 2016. Seventy-six (76) isolates of P. aeruginosa with diminished sensitivity to carbapenemes and resistant to ceftazidime were analyzed. The phenotype detection of MBL was made by double disc synergy between imipenem and meropenem versus ethylenediaminetetraacetic acid (EDTA), and the identification of the genes was performed by polymerase chain reaction. Of the 76 isolates, 24 (31.6%) were positive for MBL by the phenotype method, genetically confirming all. The blaIMP gene was found in 23/24 (95.8%) and blaVIM in 1/24 (4.2%). No isolate had blaNDM. The blaIMP gene turned out to be the most frequent among clinical isolates of P. aeruginosa not sensitive to carbapenemics at this Hospital.

Carbapenem Resistance in Gram-negative Bacteria in South-western Nigeria: The Role of Extended-spectrum lactamase CTX-M-15 / Resistencia al carbapenem en las bacterias gramnegativas en Nigeria Suroccidental: papel de la betalactamasa de espectro extendido tipo CTX-M-15

ABSTRACT Objective: To determine the role of extended-spectrum β-lactamases in carbapenem-resistant Gram-negative bacteria from south-western Nigeria. Methods: Twenty-seven carbapenem-resistant isolates that were found to be non-carbapenemase producers (15 Escherichia coli, 9 Klebsiella pneumoniae and 3 Pseudomonas aeruginosa) were further studied. These isolates were subjected to analysis including phenotypic and genotypic detection of various β-lactamases, efflux activity, outer membrane protein, plasmids replicon typing, detection of transferable genes and resistances and typing using random amplified polymorphic DNA tests. Results: No isolates demonstrated de-repression of efflux, but all showed either complete loss or reduced production of outer membrane proteins. Transconjugants from these strains contained various genes including plasmid-mediated quinolone resistance and extended-spectrum beta-lactamases. All the transconjugants carried the blaCTX-M-15 gene. The transconjugants had varying minimum inhibitory concentrations of carbapenems ranging from 0.03 μg/ml to 8 μg/ml. Varying resistances to other antimicrobial agents were also transferred with the plasmids. The donor isolates were not clonally related by molecular typing. Conclusion: Resistance to carbapenem antibiotics in this sample was not mediated only by carbapenemases but also by production of extended-spectrum β-lactamases (largely CTX-M-15), accompanied by protein loss. This was an important mechanism underpinning carbapenem resistance in these clinical isolates of various species.

RESUMEN Objetivo: Determinar el papel de las betalactamasas de espectro extendido en la resistencia al carbapenem en las bacterias gramnegativas en Nigeria. Métodos: Veintisiete aislados resistentes al carbapenem que fueron hallados productores de no carbapenemasas (15 Escherichia coli, 9 Klebsiella pneumoniae, y 3 Pseudomonas aeruginosa) fueron estudiados con mayor profundidad. Estos aislados fueron sometidos a análisis incluyendo la detección fenotípica y genotípica de varias betalactamasas, la actividad de eflujo, las porinas de la membrana externa, la tipificación del replicón plasmídico, la detección de genes transferibles y resistencias y tipificación usando pruebas de ADN polimórficas amplificadas aleatorias. Resultados: Ninguno de los aislamientos mostró desrepresión de eflujo, pero todos demostraron la pérdida completa o la producción reducida de porinas externas de la membrana. Los transconjugantes de estas cepas contenían varios genes incluyendo resistencia a la quinolona mediada por plásmidos y betalactamasas de espectro extendido. Todos los transconjugantes portaban el gen blaCTX-M-15. Los transconjugantes tenían diversas concentraciones inhibitorias mínimas de carbapenemas que oscilaban entre 0.03 μg/ml y 8 μg/ml. Varias resistencias a otros agentes antimicrobianos fueron también transferidas con los plásmidos. Los aislamientos del donante no estuvieron relacionados clonalmente por tipificación molecular. Conclusión: La resistencia al antibiótico carbapenem en esta muestra no fue mediada solamente por las carbapenemasas, sino también por la producción de betalactamasas de espectro extendido (en gran parte CTX-M-15), acompañado por pérdida de porina. Éste era un mecanismo importante que sustentaba la resistencia al carbapenem en estos aislados clínicos de varias especies.

Phenotypic and molecular detection of metallo-ß-lactamase-producing Pseudomonas aeruginosa isolates from patients with burns in Tehran, Iran

Abstract INTRODUCTION: Health care-associated infections caused by metallo-β-lactamase (MBL)-producing Pseudomonas aeruginosa are a significant growing concern in patients with burns worldwide. The aims of this study were to determine the antibiotic susceptibility of and detect the presence of MBLs among P. aeruginosa isolates and assess their clonal relationship using enterobacterial repetitive intergenic consensus (ERIC)-PCR. METHODS: Non-duplicated clinical isolates (160) of P. aeruginosa were collected from patients with burns at the Motahari Hospital in Tehran, Iran. All isolates were identified using standard laboratory methods and further characterized for antimicrobial susceptibility. Any carbapenem-resistant isolates were then examined for MBL production by the E-test and MBL-encoding genes were detected by PCR. The clonal relatedness of MBL-producing isolates was assessed by ERIC-PCR. RESULTS: For multidrug-resistant isolates, the highest rates of susceptibility were observed for colistin 160 (100%), polymyxin B 160 (100%), and ceftazidime 32 (20%). In total, 69 (43.7%) isolates were identified as MBL producers. Twenty-eight (17.5%) isolates were positive for the bla VIM-1 gene followed by the bla IMP-1 (15.6%) and bla SPM-1 (5.6%) genes. ERIC-PCR revealed three separate genotypes, where type A (76.8%) was the most prevalent, followed by B (20.3%), and then C (2.9%). CONCLUSIONS: Our present study found that the bla IMP-1 and bla VIM-1 genes were present at a significant frequency and also detected the bla SPM-1 gene in P. aeruginosa isolates for the first time, highlighting the need for establishing suitable infection control measures to successfully treat patients and prevent further spread of these resistant organisms among patients with burns.

Prevalence of metallo-ß-lactamase-encoding genes among carbapenem-resistant pseudomonas aeruginosa strains isolated from burn patients in Iran

Abstract Carbapenem-resistant Pseudomonas aeruginosa (CRPA) has been considered a major cause of infection and mortality in burn patients, especially in developing countries such as Iran. One of the most common mechanisms of carbapenem resistance is production of metallo-β-lactamases [(MBLs), including Verona Integron-encoded Metallo-beta-lactamase (VIM), imipenemase (IMP), São Paulo metalo-beta-lactamase (SPM), German imipenemase (GIM), New Delhi metallo-beta-lactamase (NDM), Dutch imipenemase (DIM), Adelaide imipenemase (AIM), Seoul imipenemase (SIM), KHM, Serratia metallo-β-lactamase (SMB), Tripoli metallo-β-lactamase (TMB), and Florence imipenemase (FIM)]. Limited information is available on the prevalence of CRPA and MBLs in Iranian burn units. We performed a systematic search by using different electronic databases, including Medline (via PubMed), Embase, Web of Science, and Iranian Database. Of 586 articles published from January 2000 to December 2016, 14 studies reporting the incidence of CRPA and MBLs as detected by molecular methods in burn patients were included in this review. The meta-analyses showed that the prevalence of CRPA, IMP, and VIM was 76.8% (95% CI 67.5-84.1), 13.1% (95% CI 4.7-31.5), and 21.4% (95% CI 14.6-30.1), respectively, in Iranian burn centers and remaining MBLs types have not yet been detected. There was a high prevalence of MBLs and CRPA in Iranian burn centers. Therefore, these measurements should be applied nationally and rigorous infection control measures and antimicrobial stewardship will be the major pillars to control multidrug resistant microorganisms, such as CRPA.

Detection of multidrug-resistant Pseudomonas aeruginosa harboring bla GES-1 and bla GES-11 in Recife, Brazil

Abstract INTRODUCTION: Pseudomonas aeruginosa, an important pathogen globally, presents several resistance mechanisms. This study aimed to investigate the presence of bla GES in clinical isolates of Pseudomonas aeruginosa obtained from various clinical specimens from patients admitted to three different hospitals in Recife, Brazil. The Guiana extended spectrum beta-lactamase (GES) enzymes are responsible for conferring broad spectrum resistance to beta-lactam drugs, including the carbapenems. METHODS: A total of 100 carbapenem-resistant P. aeruginosa isolates underwent polymerase chain reaction (PCR) testing to identify bla GES, bla KPC, bla SPM-1, bla IMP, and bla VIM. Additionally, PCR products positive for bla GES were sequenced. The clonal profiles of these same isolates were then determined by means of enterobacterial repetitive intergenic consensus (ERIC)-PCR analysis. RESULTS: PCR analysis revealed that four isolates harbored bla GES; DNA sequencing showed that two harbored bla GES-1 and two bla GES-11. Beta-lactamase genes bla SPM-1, bla IMP, bla VIM, and bla KPC were investigated; none of these genes was detected. Automated susceptibility testing methods (Vitek®2, bioMérieux) showed that the bla GES-1-positive isolates were only susceptible to polymyxin B. The patterns obtained with ERIC-PCR methods showed clonal relationship between the two isolates that harbored bla GES-11, whereas different clonal profiles were found in the isolates harboring bla GES-1. CONCLUSIONS: We detected the presence of bacterial isolates positive for two different variants of the enzyme GES in three different hospitals from Recife, Brazil. These enzymes have a great capacity for dissemination among Gram-negative bacteria and confer broad-spectrum resistance to beta-lactam antibiotics and to the carbapenems.

Carbapenem-resistant Pseudomonas aeruginosa: association with virulence genes and biofilm formation

Abstract Pseudomonas aeruginosa is an opportunistic pathogen that causes frequently nosocomial infections, currently becoming more difficult to treat due to the various resistance mechanisms and different virulence factors. The purpose of this study was to determine the risk factors independently associated with the development of bacteremia by carbapenem-resistant P. aeruginosa, the frequency of virulence genes in metallo-β-lactamases producers and to evaluate their ability to produce biofilm. We conducted a case–control study in the Uberlândia Federal University – Hospital Clinic, Brazil. Polymerase Chain Reaction was performed for metallo-β-lactamases and virulence genes. Adhesion and biofilm assays were done by quantitative tests. Among the 157 strains analyzed, 73.9% were multidrug-resistant, 43.9% were resistant to carbapenems, 16.1% were phenotypically positive for metallo-β-lactamases, and of these, 10.7% were positive for blaSPM gene and 5.3% positive for blaVIM. The multivariable analysis showed that mechanical ventilation, enteral/nasogastric tubes, primary bacteremia with unknown focus, and inappropriate therapy were independent risk factors associated with bacteremia. All tested strains were characterized as strongly biofilm producers. A higher mortality was found among patients with bacteremia by carbapenem-resistant P. aeruginosa strains, associated independently with extrinsic risk factors, however it was not evident the association with the presence of virulence and metallo-β-lactamases genes.

Distribution of the bla OXA, bla VEB-1, and bla GES-1 genes and resistance patterns of ESBL-producing Pseudomonas aeruginosa isolated from hospitals in Tehran and Qazvin, Iran

Abstract INTRODUCTION: Pseudomonas aeruginosa is one of the most common nosocomial pathogens. The emergence of extended spectrum β-lactamases (ESBLs) has been increasingly reported as a major clinical concern worldwide. The main aim of the present study was to determine the distribution of bla OXA, bla PER-1, bla VEB-1, and bla GES-1 genes among ESBL-producing P. aeruginosa isolated from two distinct provinces in Iran. METHODS: In this study, a total of 75 (27.5%) ESBL-producing isolates were identified from 273 P. aeruginosa isolates collected from patients in Qazvin and Tehran. Phenotypic detection of ESBLs and antimicrobial susceptibility testing were performed according to the Clinical and Laboratory Standards Institute guidelines. PCR and sequencing were employed to detect bla OXA-1, bla OXA, bla GES-1, bla PER-1, and bla VEB-1 genes. Isolate genetic relationships were evaluated by repetitive extragenic palindromic sequence-based PCR (REP-PCR). RESULTS: In total, 59 (78.7%) of the ESBL-producing isolates showed multidrug resistance. The highest rates of susceptibility were observed against colistin (75 isolates, 100%) and polymyxin B (75, 100%) followed by amikacin (44, 58.7%), and piperacillin-tazobactam (40, 53.3%). The bla OXA-1 (37.3%) gene was the most common of the genes investigated, followed by bla OXA-4 (32%), bla GES-1 (16%), and bla VEB-1 (13.3%). REP-PCR identified three different genotypes: types A (89.3%), B (6.7%), and C (4%). CONCLUSIONS: We found a significant presence of bla OXA-1, bla OXA-4, bla GES-1, and bla VEB-1 genes among P. aeruginosa isolates, highlighting the need for suitable infection control strategies to effectively treat patients and prevent the further distribution of these resistant organisms.

Identification of São Paulo metallo-beta-lactamase-1-producing Pseudomonas aeruginosa in the Central-West region of Brazil: a case study

Abstract: Metallo-beta-lactamase production is an important mechanism for carbapenem resistance of Pseudomonas aeruginosa , which represents an emerging public health challenge. We report the case of a patient admitted to an intensive care unit, with sepsis caused by multidrug-resistant São Paulo Metallo-beta-lactamase-1-producing P. aeruginosa . This is the first case of infection by this pathogenic strain in the State of Mato Grosso do Sul, Brazil. Thus, infection control measures are required for preventing future spread and outbreaks.

Carbapenem-resistant and cephalosporin-susceptible: a worrisome phenotype among Pseudomonas aeruginosa clinical isolates in Brazil

Abstract The mechanisms involved in the uncommon resistance phenotype, carbapenem resistance and broad-spectrum cephalosporin susceptibility, were investigated in 25 Pseudomonas aeruginosa clinical isolates that exhibited this phenotype, which were recovered from three different hospitals located in São Paulo, Brazil. The antimicrobial susceptibility profile was determined by CLSI broth microdilution. β-lactamase-encoding genes were investigated by PCR followed by DNA sequencing. Carbapenem hydrolysis activity was investigated by spectrophotometer and MALDI-TOF assays. The mRNA transcription level of oprD was assessed by qRT-PCR and the outer membrane proteins profile was evaluated by SDS-PAGE. Genetic relationship among P. aeruginosa isolates was assessed by PFGE. Carbapenems hydrolysis was not detected by carbapenemase assay in the carbapenem-resistant and cephalosporin-susceptible P. aueruginosa clinical isolates. OprD decreased expression was observed in all P. aeruginosa isolates by qRT-PCR. The outer membrane protein profile by SDS-PAGE suggested a change in the expression of the 46 kDa porin that could correspond to OprD porin. The isolates were clustered into 17 genotypes without predominance of a specific PFGE pattern. These results emphasize the involvement of multiple chromosomal mechanisms in carbapenem-resistance among clinical isolates of P. aeruginosa, alert for adaptation of P. aeruginosa clinical isolates under antimicrobial selective pressure and make aware of the emergence of an uncommon phenotype among P. aeruginosa clinical isolates.

Mechanisms of carbapenem resistance in endemic Pseudomonas aeruginosa isolates after an SPM-1 metallo-β -lactamase producing strain subsided in an intensive care unit of a teaching hospital in Brazil

Carbapenem-resistance mechanisms are a challenge in the treatment of Pseudomonas aeruginosa infections. We investigated changes in P. aeruginosa carbapenem-resistance determinants over a time period of eight years after the emergence of São Paulo metallo-β-lactamase in a university hospital in Rio de Janeiro, Brazil. Patients admitted to the intensive care unit (ICU) were screened for P. aeruginosa colonisation and followed for the occurrence of infections from April 2007 to April 2008. The ICU environment was also sampled. Isolates were typed using random amplified polymorphic DNA, pulsed-field gel electrophoresis and multilocus sequence typing. Antimicrobial susceptibility was determined by disk diffusion and E-test, production of carbapenemases by a modified-CarbaNP test and presence of carbapenemase-encoding genes by polymerase chain reaction. Non-carbapenemase resistance mechanisms studied included efflux and AmpC overexpression by PAβN and cloxacillin susceptibility enhancement, respectively, as well as oprD mutations. From 472 P. aeruginosa clinical isolates (93 patients) and 17 isolates from the ICU environment, high genotypic diversity and several international clones were observed; one environment isolate belonged to the blaSPM-1 P. aeruginosa epidemic genotype. Among isolates from infections, 10 (29%) were carbapenem resistant: none produced carbapenemases, three exhibited all non-carbapenemase mechanisms studied, six presented a combination of two mechanisms, and one exclusively displayed oprD mutations. Carbapenem-resistant P. aeruginosa displayed a polyclonal profile after the SPM-1 epidemic genotype declined. This phenomenon is connected with blaSPM-1 P. aeruginosa replaced by other carbapenem-resistant pathogens.

Clinical and microbiological features of infections caused by Pseudomonas aeruginosa in patients hospitalized in intensive care units

Abstract: INTRODUCTION: The spread of multidrug-resistant Pseudomonas aeruginosa in Brazilian hospitals has greatly impacted upon the morbidity and mortality of individuals in intensive care units. Given the lack of information regarding the dynamics of multidrug resistance in northern Brazil, we analyzed the clinical and microbiological features of nosocomial infections caused by P. aeruginosa. METHODS Between January 2010 and March 2012, we conducted a retrospective cohort study of P. aeruginosa isolates from 54 patients who were hospitalized in intensive care units. The clinical and epidemiologic variables were analyzed, including the patients' demographic data and comorbidities, and the lengths of the intensive care unit stays, the classification of the infections as nosocomial, the use of invasive procedures, antimicrobial therapy, and the patients' outcomes. We undertook susceptibility tests, molecular detection of the metallo-β-lactamase genes, and genotypic analyses of the isolates using the repetitive element-polymerase chain reaction. RESULTS: Multidrug resistance occurred most frequently among isolates from adults who had been hospitalized for an average of 87.1 days. The use of mechanical ventilation and urinary catheters were risk factors for infection. The four isolates that harbored the blaSPM-1-like gene showed >95% genetic similarity. CONCLUSIONS This study's findings show that P. aeruginosa has a high death rate, and that inadequate treatment and invasive procedures are risk factors for infection. This is the first report describing the detection of the blaSPM-1-like gene in northern Brazil. These results highlight the need for better monitoring and a greater understanding of nosocomial infections and their public health impacts.

Mutational and acquired carbapenem resistance mechanisms in multidrug resistant Pseudomonas aeruginosa clinical isolates from Recife, Brazil

An investigation was carried out into the genetic mechanisms responsible for multidrug resistance in nine carbapenem-resistant Pseudomonas aeruginosaisolates from different hospitals in Recife, Brazil. Susceptibility to antimicrobial agents was determined by broth microdilution. Polymerase chain reaction (PCR) was employed to detect the presence of genes encoding β-lactamases, aminoglycoside-modifying enzymes (AMEs), 16S rRNA methylases, integron-related genes and OprD. Expression of genes coding for efflux pumps and AmpC cephalosporinase were assessed by quantitative PCR. The outer membrane proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The blaSPM-1, blaKPC-2 and blaGES-1 genes were detected in P. aeruginosaisolates in addition to different AME genes. The loss of OprD in nine isolates was mainly due to frameshift mutations, premature stop codons and point mutations. An association of loss of OprD with the overexpression of MexAB-OprM and MexXY-OprM was observed in most isolates. Hyper-production of AmpC was also observed in three isolates. Clonal relationship of the isolates was determined by repetitive element palindromic-PCR and multilocus sequence typing. Our results show that the loss of OprD along with overexpression of efflux pumps and β-lactamase production were responsible for the multidrug resistance in the isolates analysed.

Multiple antibiotic susceptibility of polyphosphate kinase mutants (ppk1 and ppk2) from Pseudomonas aeruginosa PAO1 as revealed by global phenotypic analysis

BACKGROUND: Pseudomonas aeruginosa is known to be a multidrug resistant opportunistic pathogen. Particularly, P. aeruginosa PAO1 polyphosphate kinase mutant (ppk1) is deficient in motility, quorum sensing, biofilm formation and virulence. FINDINGS: By using Phenotypic Microarrays (PM) we analyzed near 2000 phenotypes of P. aeruginosa PAO1 polyP kinase mutants (ppk1 and ppk2). We found that both ppk mutants shared most of the phenotypic changes and interestingly many of them related to susceptibility toward numerous and different type of antibiotics such as Ciprofloxacin, Chloramphenicol and Rifampicin. CONCLUSIONS: Combining the fact that ppk1 mutants have reduced virulence and are more susceptible to antibiotics, polyP synthesis and particularly PPK1, is a good target for the design of molecules with anti-virulence and anti-persistence properties.

NDM-1 (New Delhi metallo beta lactamase-1) producing Gram-negative bacilli: Emergence & clinical implications.

Backgound & objectives: resistance to carbapenems in Gram-negative bacteria conferred by NDM-1 is a global health problem. We investigated the occurrence of NDM-1 in clinical isolates of gram-negative bacilli in a tertiary care hospital in Kashmir valley, India. Methods: Gram-negative bacilli from different clinical isolates were included in the study. Antimicrobial susceptibility was performed by Kirby Bauer disk diffusion method and interpreted using Clinical Laboratory Standards Institute (CLSI) guidelines. Isolates resistant to carbapenems were subjected to different phenotypic test such as modified hodge test (MHT), boronic acid and oxacillin based MHT (bA-MHT and OXA-MHT), combined disk test and minimum inhibitory concentration (MIC) with imipenem and imipenem -EDTA for determination of class B metallo enzymes. Presence of blaNDM-1 gene was established by PCR and confirmed by sequencing. Results: Of the total 1625 gram-negative isolates received, 100 were resistant to imipenem. Of the 100 isolates, 55 (55%) were positive by modified Hodge test indicating carbapenemase production. Of the 100 isolates tested by MHT, BA-MHT and OXA-MHT, 29 (29%) isolates belonged to Class A and 15 (15%) to Class B, while 56 (56%) isolates were negative. Of the 15 class B metallo beta lactamase producers, nine carried the blaNDM-1 gene. NDM-1 was found among escherichia coli (2 isolates), Klebsiella pneumoniae (2 isolates), Citrobacter freundii (3 isolates), Acinetobacter spp (1 isolate), and one isolate of Pseudomonas aeruginosa. Isolates were resistant to all antibiotic tested except polymyxin B and tigecycline. Interpretation & conclusions: Our study showed the presence of clinical isolates expressing NDM-1 in Srinagar, Jammu & Kashmir, India. These isolates harbour plasmid mediated multiple drug resistant determinants and can disseminate easily across several unrelated genera. To halt their spread, early identification of these isolates is mandatory.

Evaluation of tests to predict metallo-β-lactamase in cystic fibrosis (CF) and non-(CF) Pseudomonas

Double disks synergy test (DDST) and combined disks test (CD) were evaluated to predict the presence of metallo-β-lactamase in 70 Pseudomonas aeruginosa isolates recovered from cystic fibrosis and non-cystic fibrosis patients. DDST CAZ-EDTA 1 cm and CD IMP-EDTA tests showed the best accuracy (94.3%). Furthermore, for other combinations, accuracy unsatisfactory was obtained.

Evolución de la resistencia antimicrobiana de bacilos Gram negativos en unidades de cuidados intensivos en Colombia / Evolution of antimicrobial resistance in Gram negative bacilli from intensive care units in Colombia

Introducción. La evolución de la resistencia bacteriana constituye una amenaza para la salud pública mundial. Los sistemas de vigilancia epidemiológica han integrado técnicas de biología molecular para mejorar las estrategias de control. Objetivo. Describir los perfiles moleculares y fenotípicos de los bacilos Gram negativos en unidades de cuidados intensivos de 23 hospitales de Colombia entre 2009 y 2012. Materiales y métodos. Se diseñó un estudio descriptivo en 23 hospitales del Grupo para el Estudio de la Resistencia Nosocomial (sic.) en Colombia. Se analizaron 38.048 aislamientos usando WHONET durante el periodo descrito. Se describieron perfiles de resistencia para Escherichia coli , Klebsiella pneumoniae , Pseudomonas aeruginosa y Acinetobacter baumannii. En 1.248 cepas se realizó reacción en cadena de la polimerasa (PCR) para detectar las carbapenemasas clínicamente más relevantes. Resultados. Escherichia coli fue el microorganismo más frecuente (promedio=14,8 %); la frecuencia de aislamientos de K. pneumoniae aumentó de 11 % en 2009 a 15 % en 2012 (p<0,001). La tendencia de los perfiles de multirresistencia aumentó en todas las especies estudiadas. De los aislamientos de K. pneumoniae evaluados, 68,4 % fue positivo para KPC ( Klebsiella pneumoniae Carbapenemase ), mientras que la VIM ( Verona Integron-encoded Metallo-betalactamase ) en P. aeruginosa se observó en 46,5 %. Conclusiones. Se observó un incremento en la tendencia de los microorganismos hacia la multirresistencia y una amplia distribución de las carbapenemasas. La articulación de la biología molecular con los sistemas de vigilancia permitió integrar el análisis del fenotipo con los mecanismos de resistencia involucrados en las bacterias estudiadas. Este análisis permitirá la elaboración de guías para el uso adecuado de antimicrobianos y contribuirá a la contención de estas bacterias multirresistentes en Colombia.

Introduction: The continuous evolution of antimicrobial resistance poses a major threat to public health worldwide. Molecular biology techniques have been integrated to epidemiological surveillance systems to improve the control strategies of this phenomenon. Objective: To describe the phenotypic and molecular profiles of the most important Gram negative bacilli from intensive care units in 23 Colombian hospitals during the study period 2009-2012. Materials and methods: A descriptive study was conducted in 23 hospitals belonging to the Colombian Nosocomial Resistance Study Group. A total of 38.048 bacterial isolates were analyzed using WHONET over a four-year period. The antimicrobial resistant profiles were described for Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa, and Acinetobacter baumannii . Polymerase chain reaction was performed in 1.248 strains to detect the most clinically relevant carbapenemases. Results: Escherichia coli was the most frequently isolated organism (mean=14.8%). Frequency of K. pneumoniae increased significantly from 11% in 2009 to 15% in 2012 (p<0.001). All screened isolates had rising trends of multidrug-resistant profiles. KPC ( Klebsiella pneumoniae carbapenemase) was detected in 68.4% of K. pneumoniae isolates while VIM (Verona integron-encoded metallo-betalactamase) was present in 46.5% of them. Conclusion: In this study, an increase in the trend of multidrug-resistant organisms and a wide distribution of carbapenemases was observed. The integration of molecular biology to surveillance systems allowed the compilation of this data, which will aid in the construction of guidelines on antimicrobial stewardship for prevention in Colombia.