Antimicrobial use and incidence of multidrug-resistant Pseudomonas aeruginosa in a teaching hospital: an ecological approach

INTRODUCTION: Multidrug-resistant Pseudomonas aeruginosa is a major threat in healthcare settings. The use of antimicrobials can influence the incidence of resistant strains by direct and indirect mechanisms. The latter can be addressed by ecological studies. METHODS: Our group attempted to analyze the relation between the use of antipseudomonal drugs and the incidence of MDR-PA among 18 units from a 400-bed teaching hospital. The study had a retrospective, ecological design, comprising data from 2004 and 2005. Data on the use of four antimicrobials (amikacin, ciprofloxacin, ceftazidime and imipenem) were tested for correlation with the incidence of MDR-PA (defined as isolates resistant to the four antimicrobials of interest) in clinical cultures. Univariate and multivariate linear regression analyses were performed. RESULTS: Significant correlations were determined between use and resistance for all antimicrobials in the univariate analysis: amikacin (standardized correlation coefficient = 0.73, p = 0.001); ciprofloxacin (0.71, p = 0.001); ceftazidime (0.61, p = 0.007) and imipenem (0.87, p < 0.001). In multivariate analysis, only imipenem (0.67, p = 0.01) was independently related to the incidence of multidrug-resistant strains. CONCLUSIONS: These findings share similarities with those reported in individual-based observational studies, with possible implications for infection control.

Ocorrência e perfil de sensibilidade a antimicrobianos em Pseudomonas aeruginosa e Acinetobacter sp. em um hospital terciário, no sul do Brasil

INTRODUÇÃO: O principal mecanismo de resistência entre isolados de Pseudomonas aeruginosa e Acinetobacter sp. é a produção de metalo-β-lactamases (MβLs). As MβLs são enzimas capazes de hidrolisar cefalosporinas, penicilinas e carbapenêmicos, mas não monobactâmicos (aztreonam) antibióticos que se encontram entre as principais opções terapêuticas para o tratamento de infecções causadas por bactérias não fermentadoras de glicose. MÉTODOS: Um estudo observacional, transversal, descritivo e retrospectivo foi desenvolvido para avaliar a frequência e o perfil de susceptibilidade cepas de P. aeruginosa e Acinetobacter sp. produtoras de MβLs isoladas no Hospital São Vicente de Paulo, Passo Fundo, Brasil. RESULTADOS: A produção de MβLs foi observada em 77,6% (n = 173/223) dos isolados de P. aeruginosa e em 22,4% (n = 50/223) dos isolados de Acinetobacter sp. Dentre as cepas produtoras de MβL, a maioria apresentou mais de 90% de resistência a seis antimicrobianos dos 12 testados, enfatizando a resistência a ceftazidima, gentamicina, aztreonam, piperaciclina/tazobactam, cefepime, ciprofloxacina, meropenem e tobramicina. CONCLUSÕES: Os índices de MβL encontrados confirmam a preocupação mundial com a disseminação desse mecanismo de resistência.

Prevalence and clinical outcomes of episodes of ventilator-associated pneumonia caused by SPM-1-producing and non-producing imipenem-resistant Pseudomonas aeruginosa

INTRODUCTION: Pseudomonas aeruginosa is a leading cause of ventilator-associated pneumonia (VAP) and exhibits high rates of resistance to several antimicrobial drugs. The carbapenens are usually the drugs of choice against this microorganism. However, the carbapenem resistance has increased among these strains worldwide. The presence of metallo-β-lactamases (MBL) has been pointed out as a major mechanism of resistance among these strains. No previous study addressed outcomes of respiratory infections caused by these strains. METHODS: Our group sought to analyze the epidemiology and clinical outcomes of patients with VAP caused by imipenem-resistant P. aeruginosa. A total of 29 clinical isolates of carbapenem-resistant Pseudomonas aeruginosa were screened for metallo-β-lactamase (MBL) genes. RESULTS: Demographic and clinical variables were similar between the SPM-1-producing and non-SPM-1-producing group. Five (17.2%) isolates were positive for blaSPM-1. No other MBL gene was found. All patients were treated with polymyxin B. The infection-related mortality was 40% and 54.2% for SPM-1-producing and -non-producing isolates, respectively. CONCLUSIONS: There were no differences in epidemiological and clinical outcomes between the two groups.

Phenotypic and molecular characterization of antimicrobial resistance and virulence factors in Pseudomonas aeruginosa clinical isolates from Recife, State of Pernambuco, Brazil

INTRODUCTION: The emergence of carbapenem resistance mechanisms in Pseudomonas aeruginosa has been outstanding due to the wide spectrum of antimicrobial degradation of these bacteria, reducing of therapeutic options. METHODS: Sixty-one clinical strains of P. aeruginosa isolated from five public hospitals in Recife, Pernambuco, Brazil, were examined between 2006 and 2010, aiming of evaluating the profiles of virulence, resistance to antimicrobials, presence of metallo-β-lactamase (MBL) genes, and clonal relationship among isolates. RESULTS: A high percentage of virulence factors (34.4% mucoid colonies; 70.5% pyocyanin; 93.4% gelatinase positives; and 72.1% hemolysin positive) and a high percentage of antimicrobial resistance rates (4.9% pan-resistant and 54.1% multi-drug resistant isolates) were observed. Among the 29 isolates resistant to imipenem and/or ceftazidime, 44.8% (13/29) were MBL producers by phenotypic evaluation, and of these, 46.2% (6/13) were positive for the blaSPM-1 gene. The blaIMP and blaVIM genes were not detected. The molecular typing revealed 21 molecular profiles of which seven were detected in distinct hospitals and periods. Among the six positive blaSPM-1 isolates, three presented the same clonal profile and were from the same hospital, whereas the other three presented different clonal profiles. CONCLUSIONS: These results revealed that P. aeruginosa is able to accumulate different resistance and virulence factors, making the treatment of infections difficult. The identification of blaSPM-1 genes and the dissemination of clones in different hospitals, indicate the need for stricter application of infection control measures in hospitals in Recife, Brazil, aiming at reducing costs and damages caused by P. aeruginosa infections.

In vitro activity of antimicrobial combinations against multidrug-resistant Pseudomonas aeruginosa

Introduction Pseudomonas aeruginosa isolates related to nosocomial infections are often resistant to multiple antibacterial agents. In this study, antimicrobial combinations were evaluated to detect in vitro synergy against clinical isolates of P. aeruginosa. Methods Four clinical P. aeruginosa isolates were selected at random among other isolates from inpatients treated at the public University hospital in Ribeirão Preto, SP, Brazil. Two isolates were susceptible to imipenem (IPM-S) and several other antimicrobials, while the other two isolates were imipenem and multidrug resistant (IPM-R). The checkerboard method was used to assess the interactions between antimicrobials. Results Combinations of imipenem or other anti-Pseudomonas drugs with complementary antibiotics, such as aminoglycosides, fosfomycin and rifampin, reached synergy rates of 20.8%, 50%, 62.5% and 50% for the two IPM-S and two IPM-R Pseudomonas isolates, respectively. Imipenem, piperacillin-tazobactam and ceftazidime yielded a greater synergy rate than cefepime or ciprofloxacin. Synergist combinations were more commonly observed when the complementary drug was tobramycin (65%) or fosfomycin (57%). Conclusions Some antibacterial combinations led to significant reductions of the minimum inhibitory concentrations of both drugs, suggesting that they could be clinically applied to control infections caused by multidrug-resistant P. aeruginosa.

Phenotypic detection of metallo-β-lactamases in Pseudomonas aeruginosa and Acinetobacter baumannii isolated from hospitalized patients in São Luis, State of Maranhão, Brazil

Introduction Acquired metallo-β-lactamases (MβL) are emerging determinants of resistance in Pseudomonas aeruginosa and Acinetobacter baumannii. The objectives of this study were to phenotypically detect MβL in imipenem-resistant P. aeruginosa and A. baumannii, to investigate the association between MβL-positive strains and hospitals, and to compare the resistance profiles of MβL-producing and non-MβL-producing strains. Methods The approximation disk and combined disk assay methods were used in this study. Results A total of 18 (38.3%) P. aeruginosa isolates and 1 (5.6%) A. baumannii isolate tested positive for the presence of MβL. Conclusions These results demonstrate the need for strict surveillance and for the adoption of preventive measures to reduce the spread of infection and potential outbreaks of disease caused by MβL-producing microorganisms.

Production of metallo-β-lactamase among Pseudomonas aeruginosa strains isolated in the State of Sergipe, Brazil

INTRODUCTION: Acquired production of metallo-β-lactamases is an important mechanism of resistance in Pseudomonas aeruginosa. The objective of this study was to investigate the production of metallo-β-lactamase and the genetic diversity among ceftazidime-resistant P. aeruginosa isolates from State of Sergipe, Brazil. METHODS: Metallo-β-lactamase was investigated using the disk approximation test and polymerase chain reaction (PCR). Genetic diversity was evaluated by pulsed-field gel electrophoresis (PFGE). RESULTS: A total of 48 (51.6%) isolates were resistant to ceftazidime. Six (12.2%) of these were positive for metallo-β-lactamase production. Only two (4.1%) of the ceftazidime-resistant isolates carried the bla SPM-1 gene. CONCLUSIONS: Production of metallo-β-lactamases was not the main mechanism of resistance to ceftazidime and carbapenems among P. aeruginosa strains in Sergipe, Brazil.

Antibacterial, anti-swarming and anti-biofilm formation activities of Chamaemelum nobile against Pseudomonas aeruginosa

AbstractINTRODUCTION:Chamomile ( Chamaemelum nobile ) is widely used throughout the world, and has anti-inflammatory, deodorant, bacteriostatic, antimicrobial, carminative, sedative, antiseptic, anti-catarrhal, and spasmolytic properties. Because of the increasing incidence of drug-resistant bacteria, the development of natural antibacterial sources such as medical herbs for the treatment of infectious diseases is necessary. Extracts from different plant parts such as the leaves, flowers, fruit, and bark of Combretum albiflorum, Laurus nobilis , and Sonchus oleraceus were found to possess anti-quorum sensing (QS) activities. In this study, we evaluated the effect of C. nobile against Pseudomonas aeruginosa biofilm formationMETHODS:The P. aeruginosa samples were isolated from patients with different types of infection, including wound infection, septicemia, and urinary tract infection. The flowers of C. nobile were dried and the extract was removed using a rotary device and then dissolved in dimethyl sulfoxide at pH 7.4. The microdilution method was used to evaluate the minimum inhibitory concentration (MIC) of this extract on P. aeruginosa , and biofilm inhibition was assayed.RESULTS:Eighty percent of the isolated samples (16/20) could form a biofilm, and most of these were isolated from wound infections. The biofilm inhibitory concentration of the C. nobile extract was 6.25-25mg/ml, whereas the MIC was 12.5-50mg/ml.CONCLUSIONS:The anti-QS property of C. nobile may play an important role in its antibacterial activity, thus offering an additional strategy in the fight against bacterial infections. However, molecular investigation is required to explore the exact mechanisms of the antibacterial action and functions of this phytocompound.

Monoterpene isolated from the essential oil of Trachyspermum ammi is cytotoxic to multidrug-resistant Pseudomonas aeruginosa and Staphylococcus aureus strains

Abstract INTRODUCTION: The aim of this study was to determine whether an herbal extract containing monoterpene exhibited activity against multidrug-resistant Staphylococcus aureus and Pseudomonas aeruginosa isolated from clinical infection samples. METHODS: The essential oil of Trachyspermum ammi (L.) Sprague ex Turrill (Apiaceae) fruit was extracted by hydrodistillation. Fruit residues were treated with hydrochloric acid and re-hydrodistilled to obtain volatile compounds. Compounds in the distilled oil were identified using gas-chromatography (GC) and GC-mass spectrometry (MS). The antibiotic susceptibility of all bacterial isolates was analyzed using both the disc diffusion method and determination of the minimum inhibitory concentration (MIC). The sensitivity of antibiotic-resistant isolates to essential oil was also determined by using the disc diffusion method and MIC determination. RESULTS: Of 26 clinical isolates, 92% were multidrug-resistant (MDR). Aromatic monoterpenes (thymol, paracymene, and gamma-terpinene) were the major (90%) components of the oil. Growth of S. aureus strains was successfully inhibited by the oil, with an inhibitory zone diameter (IZD) between 30-60mm and MIC <0.02μL/mL. The oil had no antimicrobial activity against clinical isolates of P. aeruginosa; rather, it prevented pigment production in these isolates. CONCLUSIONS: This study revealed that the essential oil of Trachyspermum ammi, which contains monoterpene, has good antibacterial potency. Monoterpenes could thus be incorporated into antimicrobial ointment formulas in order to treat highly drug-resistant S. aureus infections. Our findings also underscore the utility of research on natural products in order to combat bacterial multidrug resistance.

Pseudomonas aeruginosa adhesion to normal and injured respiratory mucosa

Human nasal polyps outgrowth culture were used to study the adhesion of Pseudomonas aeruginosa to respiratory cells. By transmission electron microscopy, bacteria associated with ciliated cells were identified trapped at the extremities of cilia, usually as aggregates of several bacterial cells. They were never seen at the interciliary spaces or attached along cilia. Bacteria were also seen to adhere to migrating cells of the periphery of the outgrowth culture. Using a model of repair of wounded respiratory epithelial cells in culture, we observed that the adhesion of P. aeruginosa to migrating cells of the edges of the repairing wounds was significantly higher than the adhesion to non-migrating cells and that adherent bacteria were surrounded by a fibrocnectin-containing fibrillar material The secretion of extracellular matrix components is involved in the process of epithelium repair following injury. To investigate the molecular basis of P. aeruginosa adhesion to migrating cells, bacteria were treated with a fibronectin solution before their incubation with the respiratory cells. P. aeruginosa treatment by fibronectin significantly increased their adhesion to migrating cells. Accordingly, we hypothesize that during cell migration, fibronectin secreted by epithelial cells may favour P. aeruginosa adhesion by establishing a bridge between the bacteria and the epithelial cell receptors. Such a mechanism may represent a critical step for P. aeruginosa infection of healing injured epithelium.

Pseudomonas aeruginosa: study of Antibiotic Resistance and Molecular Typing in Hospital Infection Cases in a Neonatal Intensive Care Unit from Rio de Janeiro City, Brazil

This study had the objective of to analyze the demographic and bacteriologic data of 32 hospitalized newborns in an neonatal intensive care unit of a public maternity hospital in Rio de Janeiro city, Brazil, seized by Pseudomonas aeruginosa sepsis during a period ranged from July 1997 to July 1999, and to determine the antimicrobial resistance percentage, serotypes and pulsed field gel electrophoresis (PFGE) patterns of 32 strains isolated during this period. The study group presented mean age of 12.5 days, with higher prevalence of hospital infection in males (59.4%) and vaginal delivery (81.2%), than females (40.6%) and cesarean delivery (18.8%), respectively. In this group, 20 (62.5%) patients received antimicrobials before positive blood cultures presentation. A total of 87.5% of the patients were premature, 62.5% presented very low birth weight and 40.6% had asphyxia. We detected high antimicrobial resistance percentage to b-lactams, chloramphenicol, trimethoprim/sulfamethoxazole and tetracycline among the isolated strains. All isolated strains were classified as multi-drug resistant. Most strains presented serotype O11 while PFGE analysis revealed seven distinct clones with isolation predominance of a single clone (75%) isolated from July 1997 to June 1998.

Susceptibility of clinical isolates of multiresistant Pseudomonas aeruginosa to a hospital disinfectant and molecular typing

The aim of this study was to evaluate the susceptibility of 35 resistant Pseudomonas aeruginosa clinical isolates to a quaternary ammonium hospital disinfectant. The methodology was the AOAC Use-Dilution Test, with disinfectant at its use-concentration. In addition, the chromosomal DNA profile of the isolates were determined by macro-restriction pulsed field gel electrophoresis (PFGE) method aiming to verify the relatedness among them and the behavior of isolates from the same group regarding the susceptibility to the disinfectant. Seventy one percent of the isolates were multiresistant to antibiotics and 43% showed a reduced susceptibility to the disinfectant. The PFGE methodology detected 18 major clonal groups. We found isolates with reduced susceptibility to the disinfectant and we think that these are worrying data that should be further investigated including different organisms and chemical agents in order to demonstrate that microorganisms can be destroyed by biocide as necessary. We also found strains of the same clonal groups showing different susceptibility to the disinfectant. This is an interesting observation considering that only few works are available about this subject. PFGE profile seems not to be a reliable marker for resistance to disinfectants.

Emergence of dhfrXVb and blaCARB-4 gene cassettes in class 1 integrons from clinical Pseudomonas aeruginosa isolated in Amazon region

Integrons play a role in horizontal acquisition and expression of genes, as well as gene reservoir, contributing for the resistance phenotype, particularly relevant to bacteria of clinical importance. We aimed to determine the composition and the organization of the class 1 integron variable region present in Pseudomonas aeruginosa clinical isolates from Brazil. Strains carrying class 1 integrons were resistant to the majority of antibiotics tested, except to imipenem and ceftazidime. Sequence analysis of the integron variable region revealed the presence of the blaCARB-4 gene into two distinct cassette arrays: aacA4-dhfrXVb-blaCARB-4 and aadB-aacA4-blaCARB-4 . dhfrXVb gene cassette, which is rare in Brazil and in P. aeruginosa species, was found in one isolate. PFGE analysis showed the spread of blaCARB-4 among P. aeruginosa clones. The occurrence of blaCARB-4 and dhfrXVb in Brazil may contribute for developing resistance to clinically important antibiotics, and shows a diversified scenarium of these elements occurring in Amazon clinical settings, where no study about integron dinamycs was performed to date.

Changing the epidemiology of carbapenem-resistant Pseudomonas aeruginosa in a Brazilian teaching hospital: the replacement of São Paulo metallo-β-lactamase-producing isolates

In Brazil, carbapenem-resistant Pseudomonas aeruginosa isolates are closely related to the São Paulo metallo-β-lactamase (SPM) Brazilian clone. In this study, imipenem-resistant isolates were divided in two sets, 2002/2003 and 2008/2009, analysed by pulsed field gel electrophoresis and tested for the Ambler class B metallo-β-lactamase (MBL) genes blaSPM-1, blaIMP and blaVIM. The results show a prevalence of one clone related to the SPM Brazilian clone in 2002/2003. In 2008/2009, P. aeruginosa isolates were mostly MBL negative, genetically diverse and unrelated to those that had been detected earlier. These findings suggest that the resistance to carbapenems by these recent P. aeruginosa isolates was not due to the spread of MBL-positive SPM-related clones, as often observed in Brazilian hospitals.

The infection of microvascular endothelial cells with ExoU-producing Pseudomonas aeruginosa triggers the release of von Willebrand factor and platelet adhesion

An increased plasma concentration of von Willebrand factor (vWF) is detected in individuals with many infectious diseases and is accepted as a marker of endothelium activation and prothrombotic condition. To determine whether ExoU, a Pseudomonas aeruginosa cytotoxin with proinflammatory activity, enhances the release of vWF, microvascular endothelial cells were infected with the ExoU-producing PA103 P. aeruginosa strain or an exoU-deficient mutant. Significantly increased vWF concentrations were detected in conditioned medium and subendothelial extracellular matrix from cultures infected with the wild-type bacteria, as determined by enzyme-linked immunoassays. PA103-infected cells also released higher concentrations of procoagulant microparticles containing increased amounts of membrane-associated vWF, as determined by flow cytometric analyses of cell culture supernatants. Both flow cytometry and confocal microscopy showed that increased amounts of vWF were associated with cytoplasmic membranes from cells infected with the ExoU-producing bacteria. PA103-infected cultures exposed to platelet suspensions exhibited increased percentages of cells with platelet adhesion. Because no modulation of the vWF mRNA levels was detected by reverse transcription-polymerase chain reaction assays in PA103-infected cells, ExoU is likely to have induced the release of vWF from cytoplasmic stores rather than vWF gene transcription. Such release is likely to modify the thromboresistance of microvascular endothelial cells.