The protein phosphatase PPKL is a key regulator of daughter parasite development in Toxoplasma gondii.

Apicomplexan parasites, including Toxoplasma gondii, encode many plant-like proteins, which play significant roles and present attractive targets for drug development. In this study, we have characterized the plant-like protein phosphatase PPKL, which is unique to the parasite and absent in its mammalian host. We have shown that its localization changes as the parasite divides. In non-dividing parasites, it is present in the cytoplasm, nucleus, and preconoidal region. As the parasite begins division, PPKL is enriched in the preconoidal region and the cortical cytoskeleton of nascent parasites. Later in the division, PPKL is present in the basal complex ring. Conditional knockdown of PPKL showed that it is essential for parasite propagation. Moreover, parasites lacking PPKL exhibit uncoupling of division, with normal DNA duplication but severe defects in forming daughter parasites. While PPKL depletion does not impair the duplication of centrosomes, it affects the stability of cortical microtubules. Both co-immunoprecipitation and proximity labeling identified the kinase DYRK1 as a potential functional partner of PPKL. Complete knockout of DYRK1 causes parasites to exhibit division defects with predominantly asynchronous divisions. Global phosphoproteomics analysis revealed a significant increase in phosphorylation of the microtubule-associated protein SPM1 in PPKL-depleted parasites, suggesting that PPKL regulates cortical microtubules by mediating the phosphorylation state of SPM1. More importantly, the phosphorylation of cell cycle-associated kinase Crk1, a known regulator of daughter cell assembly, is altered in PPKL-depleted parasites. Thus, we propose that PPKL regulates daughter parasite development by influencing the Crk1-dependent signaling pathway. IMPORTANCE Toxoplasma gondii can cause severe disease in immunocompromised or immunosuppressed patients and during congenital infections. Treating toxoplasmosis presents enormous challenges since the parasite shares many biological processes with its mammalian hosts, which results in significant side effects with current therapies. Consequently, proteins that are essential and unique to the parasite represent favorable targets for drug development. Interestingly, Toxoplasma, like other members of the phylum Apicomplexa, has numerous plant-like proteins, many of which play crucial roles and do not have equivalents in the mammalian host. In this study, we found that the plant-like protein phosphatase PPKL appears to be a key regulator of daughter parasite development. With the depletion of PPKL, the parasite shows severe defects in forming daughter parasites. This study provides novel insights into the understanding of parasite division and offers a new potential target for the development of antiparasitic drugs.

Endemic High-Risk Clone ST277 Is Related to the Spread of SPM-1-Producing Pseudomonas aeruginosa during the COVID-19 Pandemic Period in Northern Brazil.

Pseudomonas aeruginosa is a high-priority bacterial agent that causes healthcare-acquired infections (HAIs), which often leads to serious infections and poor prognosis in vulnerable patients. Its increasing resistance to antimicrobials, associated with SPM production, is a case of public health concern. Therefore, this study aims to determine the antimicrobial resistance, virulence, and genotyping features of P. aeruginosa strains producing SPM-1 in the Northern region of Brazil. To determine the presence of virulence and resistance genes, the PCR technique was used. For the susceptibility profile of antimicrobials, the Kirby-Bauer disk diffusion method was performed on Mueller-Hinton agar. The MLST technique was used to define the ST of the isolates. The exoS+/exoU- virulotype was standard for all strains, with the aprA, lasA, toxA, exoS, exoT, and exoY genes as the most prevalent. All the isolates showed an MDR or XDR profile against the six classes of antimicrobials tested. HRC ST277 played a major role in spreading the SPM-1-producing P. aeruginosa strains.

The protein phosphatase PPKL is a key regulator of daughter parasite development in Toxoplasma gondii.

Apicomplexan parasites, including Toxoplasma gondii, encode many plant-like proteins, which play significant roles and present attractive targets for drug development. In this study, we have characterized the plant-like protein phosphatase PPKL, which is unique to the parasite and absent in its mammalian host. We have shown that its localization changes as the parasite divides. In non-dividing parasites, it is present in the cytoplasm, nucleus, and preconoidal region. As the parasite begins division, PPKL is enriched in the preconoidal region and the cortical cytoskeleton of the nascent parasites. Later in the division, PPKL is present in the basal complex ring. Conditional knockdown of PPKL showed that it is essential for parasite propagation. Moreover, parasites lacking PPKL exhibit uncoupling of division, with normal DNA duplication but severe defects in forming daughter parasites. While PPKL depletion does not impair the duplication of centrosomes, it affects the rigidity and arrangement of the cortical microtubules. Both Co-Immunoprecipitation and proximity labeling identified the kinase DYRK1 as a potential functional partner of PPKL. Complete knockout of DYRK1 phenocopies lack of PPKL, strongly suggesting a functional relationship between these two signaling proteins. Global phosphoproteomics analysis revealed a significant increase in phosphorylation of the microtubule-associated proteins SPM1 in PPKL-depleted parasites, suggesting PPKL regulates the cortical microtubules by mediating the phosphorylation state of SPM1. More importantly, the phosphorylation of cell cycle-associated kinase Crk1, a known regulator of daughter cell assembly, is altered in PPKL-depleted parasites. Thus, we propose that PPKL regulates daughter parasite development by influencing the Crk1-dependent signaling pathway.

Understanding Foot Loading and Balance Behavior of Children with Motor Sensory Processing Disorder.

Sensory processing disorder (SPD) could influence the neuromuscular response and adjustment to external sensory discrimination and lead to disruptions in daily locomotion. The objective of the current study was to compare plantar loadings and foot balance during walking, running and turning activities in SPD children in order to reveal the behavioral strategy of movement and balance control. Six SPD children and six age-match healthy controls participated in the test using a FootScan plantar pressure plate. The time-varying parameters of forces, center of pressure and foot balance index were analyzed using an open-source one-dimensional Statistical Parametric Mapping (SPM1d) package. No difference was found in foot balance and plantar loadings during walking, while limited supination-pronation motion was observed in the SPD children during running and turning. The plantar forces were mainly located in the midfoot region while less toe activity was found as well. Findings should be noted that SPD children had limited supination-pronation movement for shock attenuation in the foot complex and reduced ankle pronation to assist push-off and toe gripping movements. Understanding the behavior of plantar loading strategy and balance control during walking, running and turning activities may provide clinical implications for the rehabilitation and training of daily tasks.

Novice Female Exercisers Exhibited Different Biomechanical Loading Profiles during Full-Squat and Half-Squat Practice.

BACKGROUND: Females with different practice experience may show different body postures and movement patterns while squatting in different depths, which may lead to changes of biomechanical loadings and increase the risks of injuries. METHODS: Sixteen novice female participants without squat training experience participated in this study. A 3D motion capture system was used to collect the marker trajectory and ground reaction force data during bodyweight squatting in different depths. The participants' kinematic data and joint moment were calculated using OpenSim's inverse kinematics and inverse dynamics algorithm. In this study, authors adapted a model especially developed for squatting and customized the knee joint with extra Degree-of-Freedom (DoF) in the coronal and horizontal plane with adduction/abduction and internal/external rotation. A paired-sample t-test was used to analyze the difference of joint range of motions (ROM) and peak moments between full-squat (F-SQ) and half-squat (H-SQ). One-Dimensional Statistical Parametric Mapping (SPM1D) is used to analyze the difference of joint angle and moment between the process of squatting F-SQ and H-SQ. RESULTS: (1) Compared with H-SQ, F-SQ showed larger ROM in sagittal, coronal, and transverse planes (p < 0.05). (2) SPM1D found that the difference in joint angles and joint moments between F-SQ and H-SQ was mainly concentrated in the mid-stance during squatting, which suggested the difference is greatly pronounced during deeper squat. (3) Peak hip extension moment, knee extension moment, hip adduction moment, and plantar flexion moment of F-SQ were significantly higher than H-SQ (p < 0.05). (4) Difference of hip and knee extension moments and rotation moments between the F-SQ and H-SQ were exhibited during descending and ascending. CONCLUSIONS: The study found that novice women had larger range of joint motion during the F-SQ than H-SQ group, and knee valgus was observed during squatting to the deepest point. Greater joint moment was found during F-SQ and reached a peak during ascending after squatting to the deepest point. Novice women may have better movement control during H-SQ. The findings may provide implications for the selection of lower limb strength training programs, assist the scientific development of training movements, and provide reference for squat movement correction, thus reducing the risk of injury for novice women in squatting practice.

Primer aislado de Pseudomonas aeruginosa productora de Sao Paulo metalo-beta-lactamasa (SPM-1) en un paciente chileno / First isolate of SPM-1- producing Pseudomonas aeruginosa (Sao Paulo metallo-beta-lactamase) in a Chilean patient

Resumen Las enzimas VIM, IMP, y NDM son carbapenemasas de tipo metalo-beta-lactamasas (MBLs) que se encuentran ampliamente distribuidas en el mundo. La SPM-1 (Sao Paulo metalo-beta-lactamasa) es una MBL que fue descrita en Pseudomonas aeruginosa en Sao Paulo (Brasil) en 2002. Comunicamos por primera vez la presencia de SPM-1 en Chile, en un aislado de P aeruginosa resistente a meropenem e imipenem, detectado en un cultivo rectal de vigilancia de carbapenemasas desde un paciente internado en nuestra institución. La secuencia del producto de la RPC fue 100% idéntica a la secuencia de SPM-1 reportada en Brasil. El paciente tenía antecedentes de una angioplastía realizada en Brasil en 2004-2005. Como consecuencia de este hallazgo, la detección de SPM mediante RPC será incorporada a la búsqueda de rutina de carbapenemasas en P aeruginosa.

Abstract VIM, IMP, and NDM carbapenemases are metallo-beta-lactamases (MBLs) that are widely distributed throughout the world. SPM-1 (Sao Paulo metallo-beta-lactamase) is an MBL that was described in Pseudomonas aeruginosa in Sao Paulo (Brazil) in 2002. We report for the first time the presence of SPM-1 in Chile, in an isolate of P aeruginosa resistant to meropenem and imipenem, detected in a carbapenemase surveillance rectal swab culture, in a patient admitted to our institution. The sequence of the PCR product was 100% identical to the sequence of SPM-1 reported in Brazil. The patient had a history of an angioplasty performed in Brazil in 2004-2005. As a consequence of this finding, the detection of SPM by PCR will be incorporated into the routine screening for carbapenemases in P aeruginosa.

The Resistome of Low-Impacted Marine Environments Is Composed by Distant Metallo-ß-Lactamases Homologs.

The worldwide dispersion and sudden emergence of new antibiotic resistance genes (ARGs) determined the need in uncovering which environment participate most as their source and reservoir. ARGs closely related to those currently found in human pathogens occur in the resistome of anthropogenic impacted environments. However, the role of pristine environment as the origin and source of ARGs remains underexplored and controversy, particularly, the marine environments represented by the oceans. Here, due to the ocean nature, we hypothesized that the resistome of this pristine/low-impacted marine environment is represented by distant ARG homologs. To test this hypothesis we performed an in silico analysis on the Global Ocean Sampling (GOS) metagenomic project dataset focusing on the metallo-ß-lactamases (MßLs) as the ARG model. MßLs have been a challenge to public health, since they hydrolyze the carbapenems, one of the last therapeutic choice in clinics. Using Hidden Markov Model (HMM) profiles, we were successful in identifying a high diversity of distant MßL homologs, related to the B1, B2, and B3 subclasses. The majority of them were distributed across the Atlantic, Indian, and Pacific Oceans being related to the chromosomally encoded MßL GOB present in Elizabethkingia genus. It was observed only a reduced number of metagenomic sequence homologs related to the acquired MßL enzymes (VIM, SPM-1, and AIM-1) that currently have impact in clinics. Therefore, low antibiotic impacted marine environment, as the ocean, are unlikely the source of ARGs that have been causing enormous threat to the public health.

Shaping Substrate Selectivity in a Broad-Spectrum Metallo-ß-Lactamase.

Metallo-ß-lactamases (MBLs) are the major group of carbapenemases produced by bacterial pathogens. The design of MBL inhibitors has been limited by, among other issues, incomplete knowledge about how these enzymes modulate substrate recognition. While most MBLs are broad-spectrum enzymes, B2 MBLs are exclusive carbapenemases. This narrower substrate profile has been attributed to a sequence insertion present in B2 enzymes that limits accessibility to the active site. In this work, we evaluate the role of sequence insertions naturally occurring in the B2 enzyme Sfh-I and in the broad-spectrum B1 enzyme SPM-1. We engineered a chimeric protein in which the sequence insertion of SPM-1 was replaced by the one present in Sfh-I. The chimeric variant is a selective cephalosporinase, revealing that the substrate profile of MBLs can be further tuned depending on the protein context. These results also show that the stable scaffold of MBLs allows a modular engineering much richer than the one observed in nature.

Molecular epidemiology of SPM-1-producing Pseudomonas aeruginosa by rep-PCR in hospitals in Parana, Brazil.

BACKGROUND: Infections caused by multidrug resistant microorganisms are a global health problem, and Pseudomonas aeruginosa is an important nosocomial pathogen, easily disseminated in the hospital environment. The aim of this study was to determine SPM-1 in P. aeruginosa strains in 30 Brazilian hospitals and the genetic similarity of isolates. METHODS: We analyzed 161 isolates of carbapenem-resistant P. aeruginosa. Imipenem/EDTA and imipenem strip were used for phenotypic detection of MBL production; and real-time polymerase chain reaction (PCR) for genetic detection. Genetic similarity was determined by rep-PCR. RESULTS: We obtained 136/161 (84.5%) isolates with positive phenotypic result for metallo-ß-lactamase (MBL) and the blaSPM-1 gene was identified in 41 isolates. There was a predominant profile (>95% of genetic similarity) in 92.7% of isolates. This predominant profile was widely disseminated in Paraná state. CONCLUSION: SPM-1 is the main MBL identified in carbapenem-resistant P. aeruginosa in Southern Brazil. The genetic similarity among some isolates suggests a clonal expansion.

Caracterização fenotípica e genotípica de Acinetobacter spp. e Pseudomonas aeruginosa produtores de carbapenemases / Phenotypic and genotypic characterization of carbapenemase-producing Acinetobacter spp. e Pseudomonas aeruginosa

A resistência bacteriana a antibióticos é um grave e crescente problema de saúde pública de âmbito mundial. O principal, e mais eficiente, mecanismo de resistência aos ß-lactâmicos em bacilos Gram-negativos é a produção de ß-lactamases, que possuem a capacidade de hidrolisar o anel ß-lactâmicos e consequentemente inativar essa classe de antibióticos. Vale ressaltar, que atualmente os antibióticos ß-lactâmicos são os mais utilizados clinicamente, particularmente em infecções graves. Dentre as ß-lactamases existentes destacam-se as carbapenemases, enzimas capazes de inativar a maioria dos antibióticos ß-lactâmicos. Uma grande preocupação é o fato dessas enzimas, em sua maioria, serem codificadas por plasmídeos, o que propicia a disseminação desses genes de resistência; portanto, é de extrema importância a realização de um rápido e efetivo monitoramento da presença de patógenos portadores desses genes de resistência, para que assim se possa prevenir a disseminação desses determinantes. Foram incluídos neste estudo 230 amostras únicas de Acinetobacter e Pseudomonas aeruginosa resistentes a imipenem detectados em pacientes internados em hospitais privados da cidade de São Paulo durante o período de fevereiro a outubro de 2013. As amostras foram avaliadas quanto à hidrólise de imipenem por espectrofotometria, quanto à presença de genes de carbapenemases por PCR e sequenciamento, e quanto à clonalidade por eletroforese em campos pulsados (PFGE) ou ERIC-PCR. Foram realizados ensaios de conjugação, transformação e sequenciamento completo de plasmídeos. Dentre as amostras de Acinetobacter spp. 80% (88) foram capazes de hidrolisar o imipenem. Dentre esses 76,1% (67) foram positivos para blaOXA-51-like, 19,3% (17) foram positivos para blaOXA-72. blaOXA-23, blaOXA-482 e blaIMP-1 foram detectados isoladamente em isolados distintos. O gene blaIMP-1 foi detectado em A. ursingii inserido em integron de classe 1 e representa a primeira descrição no Brasil. Uma nova carbapenemase OXA-482-like foi detectada em A. baumanii. Utilizando-se ERIC-PCR, observou-se uma grande diversidade de grupos clonais, com o máximo de quatro isolados por grupo. Dentre as amostras de P. aeruginosa, apenas 35,3% foram capazes de hidrolisar o imipenem. Dessas amostras, 14 possuíam o gene blaSPM-1, e isolados únicos possuíam, individualmente, os genes blaIMP, blaVIM, blaKPC-2 ou blaGES-23. O gene blaKPC-2 foi detectado inserido em contexto genético diferente dos descritos anteriormente, em plasmídeo IncU de 32 Kb, mobilizável, mas não conjugativo. Esta é a primeira descrição da sequencia completa de plasmídeo albergando o gene blaKPC-2 em P. aeruginosa no Brasil. Nas demais amostras (20) com atividade hidrolítica, não foram detectados genes de carbapenemase conhecidos, o que sugere a presença de genes de carbapenemase ainda não descritos. Em três amostras foi possível obter transformantes com plasmídeos, resistentes a carbapenêmicos. As amostras com blaSPM-1 apresentaram perfis de PFGE estreitamente relacionados. Em contraste, os perfis de PFGE das amostras com potenciais novas carbapenemases apresentaram índice de similaridade de Dice inferior ix a 80%, evidenciando grande diversidade clonal. Nossos achados evidenciam que a carbapenemase não intrínseca predominante em Acinetobacterem hospitais privados da cidade de São Paulo é OXA-72, e em hospitais privados há uma grande diversidade clonal. Em P. aeruginosa, a carbapenemase predominante é SPM-1, cuja disseminação é mediada por um único clone. Há potencialmente um número significativo de novas carbapenemases em Acinetobacter e P. aeruginosa, algumas delas mediadas por plasmídeos

Bacterial resistance to antibiotics is a serious and growing public health problem worldwide. The main and most efficient mechanism of resistance to ß-lactams in Gram-negative bacilli is the production of ß-lactamases, which have the ability to hydrolyze the ß-lactam ring and consequently inactivate this class of antibiotics. It is worth mentioning that currently ß-lactam antibiotics are the most used clinically, particularly in severe infections. Among the existing ß-lactamases, carbapenemases are capable of inactivating most ß-lactam antibiotics. A major concern is that these enzymes are mostly encoded by plasmids, which facilitates the spread of these resistance genes; therefore, it is of extreme importance to carry out a rapid and effective monitoring of the presence of pathogens bearing these resistance genes, in order to prevent the dissemination of these determinants. This study included 230 unique samples of imipenem-resistant Acinetobacterand Pseudomonas aeruginosa detected in patients hospitalized in private hospitals in the city of São Paulo during the period from February to October 2013. The samples were evaluated for the imipenem hydrolysis by spectrophotometry, the presence of carbapenemase genes by PCR and sequencing, and concerning clonality by pulsed field electrophoresis (PFGE) or ERIC-PCR. Conjugation, transformation and complete sequencing of plasmids were performed. Among Acinetobacter spp. samples, 80% (88) were able to hydrolyze imipenem. Among these, 76.1% (67) were positive for blaOXA-51-like genes and 19.3% (17) were positive for blaOXA-72. The blaOXA-23, blaOXA-482 and blaIMP-1 genes were detected alone in distinct isolates. The blaIMP-1 gene was detected in A. ursingii inserted in class 1 integron and represents the first description in Brazil. A novel OXA-482-like carbapenemase was detected in A. baumanii. Using ERIC-PCR, a great diversity of clonal groups was observed, with a maximum of four isolates per group. Among P. aeruginosa samples, only 35.3% were able to hydrolyze imipenem. Of these samples, 14 had the blaSPM-1 gene, and single isolates individually possessed the blaIMP, blaVIM, blaKPC-2 or blaGES-23 genes. The blaKPC-2 gene was found inserted in a genetic context different from those described previously, in a mobilizable, but not conjugative, 32 Kb IncU plasmid. This is the first description of the complete nucleotide sequence of a plasmid harboring the blaKPC-2 gene in P. aeruginosa in Brazil. In the remaining samples (20) with hydrolytic activity, no known carbapenemase genes were detected, suggesting the presence of carbapenemase genes not yet described. In three samples it was possible to obtain transformants with plasmids, resistant to carbapenems. Samples with blaSPM-1 showed closely related PFGE profiles. In contrast, the PFGE profiles of the samples with potential new carbapenemases showed Dice similarity index lower than 80%, evidencing a great clonal diversity. Our findings show that the predominant non-intrinsic carbapenemase in Acinetobacter in the city of São Paulo is OXA-72, and in private hospitals there is great clonal diversity. In P. aeruginosa, the predominant carbapenemase is SPM-1, the spread of this enzyme is mediated by a single clone. There are potentially a significant number of new carbapenemases in Acinetobacter and P. aeruginosa, some of them plasmid mediated

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.

Virulence attributes in Brazilian clinical isolates of Pseudomonas aeruginosa.

Pseudomonas aeruginosa is an opportunistic human pathogen responsible for causing a huge variety of acute and chronic infections with significant levels of morbidity and mortality. Its success as a pathogen comes from its genetic/metabolic plasticity, intrinsic/acquired antimicrobial resistance, capacity to form biofilm and expression of numerous virulence factors. Herein, we have analyzed the genetic variability, antimicrobial susceptibility as well as the production of metallo-ß-lactamases (MBLs) and virulence attributes (elastase, pyocyanin and biofilm) in 96 strains of P. aeruginosa isolated from different anatomical sites of patients attended at Brazilian hospitals. Our results revealed a great genetic variability, in which 86 distinct RAPD types (89.6% of polymorphisms) were detected. Regarding the susceptibility profile, 48 strains (50%) were resistant to the antimicrobials, as follows: 22.92% to the three tested antibiotics, 12.5% to both imipenem and meropenem, 11.46% to ceftazidime only, 2.08% to imipenem only and 1.04% to both ceftazidime and meropenem. Out of the 34 clinical strains of P. aeruginosa resistant to both imipenem and meropenem, 25 (73.53%) were MBL producers by phenotypic method while 12 (35.29%) were PCR positive for the MBL gene SPM-1. All P. aeruginosa strains produced pyocyanin, elastase and biofilm, although in different levels. Some associations were demonstrated among the susceptibility and/or production of these virulence traits with the anatomical site of strain isolation. For instance, almost all strains isolated from urine (85.71%) were resistant to the three antibiotics, while the vast majority of strains isolated from rectum (95%) and mouth (66.67%) were susceptible to all tested antibiotics. Urine isolates produced the highest pyocyanin concentration (20.15±5.65 µg/ml), while strains isolated from pleural secretion and mouth produced elevated elastase activity (1441.43±303.08 FAU) and biofilm formation (OD590 0.676±0.32), respectively. Also, MBL-positive strains produced robust biofilm compared to MBL-negative strains. Collectively, the production of site-dependent virulence factors can be highlighted as potential therapeutic targets for the treatment of infections caused by heterogeneous and resistant strains of P. aeruginosa.

Metallo-beta-lactamases among imipenem-resistant Pseudomonas aeruginosa in a Brazilian university hospital

INTRODUCTION: Imipenem-resistant Pseudomonas aeruginosa resulting from metallo-β-lactamases has been reported to be an important cause of nosocomial infection and is a critical therapeutic problem worldwide, especially in the case of bacteremia. OBJECTIVES: To determine the frequency of metallo-β-lactamases among imipenem-resistant Pseudomonas aeruginosa isolates and to compare methods of phenotypic and molecular detection. METHODS: During 2006, 69 imipenem-resistant Pseudomonas aeruginosa samples were isolated from blood and tested for metallo-β-lactamase production using both phenotypic methods. Minimal Inhibitory Concentratrions (MIC) (μg/mL) was determined with commercial microdilution panels. Pulsed Field Gel Electrophoresis (PFGE) was performed among metallo-β-lactamase producers. RESULTS: Of all the blood isolates, 34.5 percent were found to be imipenem-resistant Pseudomonas aeruginosa. Positive phenotypic tests for metallo-β-lactamases ranged from 28 percent-77 percent, and Polymerase Chain Reaction (PCR) were positive in 30 percent (of note, 81 percent of those samples were blaSPM-1 and 19 percent were blaVIM-2). Ethylenediamine tetracetic acid (EDTA) combinations for the detected enzymes had low kappa values; thus, care should be taken when use it as a phenotypic indicator of MBL. Despite a very resistant antibiogram, four isolates demonstrated the worrisome finding of a colistin MIC in the resistant range. PFGE showed a clonal pattern. CONCLUSION: Metallo-β-lactamases among imipenem-resistant Pseudomonas aeruginosa were detected in 30.4 percent of imipenem-resistant Pseudomonas aeruginosa isolates. This number might have been higher if other genes were included. SPM-1 was the predominant enzyme found. Phenotypic tests with low kappa values could be misleading when testing for metallo-β-lactamases. Polymerase Chain Reaction detection remains the gold standard.