Prevalence, antibiotic resistance profile, MBLs encoding genes, and biofilm formation among clinical carbapenem-resistant Enterobacterales isolated from patients in Mohammed VI University Hospital Centre, Morocco.

Enterobacterales are frequently a major cause of human infections. The emergence of carbapenem resistance as well as the biofilm formation complicate their management. In this regard, this study aimed to investigate the prevalence, antibiogram, carbapenemase genes, and biofilm production among Enterobacterales. For this purpose, 18 172 clinical specimens from hospitalized patients at Mohammed VI University Hospital were collected over two years (2018-2019). The bacteriological investigation was performed to isolate Enterobacterales. Subsequently, BD-Phoenix and MALDI-TOF-MS were used for bacterial identification. The production of ESBLs and carbapenemases was assessed using phenotypic tests and PCR. The biofilm formation was eventually carried out. Out of 195 carbapenem-resistant Enterobacterales strains, 190 were carbapenemase producers, and 74 Enterobacterales produced metallo-beta-lactamases (MBLs). The PCR results revealed that blaNDM was the most common carbapenemase gene, present in 62 cases, followed by the co-existence of blaNDM and blaOXA-48 in 12 cases. Klebsiella pneumoniae was the most frequently identified species among the 74 New Delhi metallo-ß-lactamase (NDM) isolates and the XDR resistance phenotype was the most prevalent with 58.10%. Additionally, all 74 NDM-positive Enterobacterales were able to form biofilms, with 82.4% being strong producers. This study highlights the need for rapid detection of carbapenemase and biofilm production in our hospital to manage this health concern.

An updated patent review of metallo-ß-lactamase inhibitors (2020-2023).

INTRODUCTION: Metallo-ß-lactamases (MBLs) are enzymes produced by bacteria that confer resistance to most ß-lactam antibiotics, including carbapenems, which have the broadest spectrum of activity. This resistance mechanism poses a significant threat to public health as it drastically reduces treatment options for severe bacterial infections. Developing effective inhibitors against MBLs is crucial to restore susceptibility to ß-lactam antibiotics. AREAS COVERED: This review aims to provide an updated analysis of patents describing novel MBL inhibitors and their potential therapeutic applications that were filed between January 2020 and May 2023. EXPERT OPINION: Significant advancements were made in the development of selective MBL inhibitors with zinc-binding and zinc-chelating mechanisms of action. Dual inhibitors, targeting simultaneously both serine-ß-lactamases (SBLs) and MBLs, represent an interesting alternative approach that is increasingly pertinent for the treatment of infections involving multiple ß-lactamases from different Ambler classes. Most examples of MBL-specific inhibitors were focused on the treatment of MBL-mediated infections in Enterobacterales, where IMP-1 was a more difficult target compared with VIM-1 or NDM-1, and much less on Pseudomonas aeruginosa or Acinetobacter baumannii, which are more challenging to address.

Characterization of Three Novel IMP Metallo-ß-Lactamases, IMP-89, IMP-91, and IMP-96, and Diverse blaIMP-Carrying Accessory Genetic Elements from Chinese Clinical Isolates.

Three novel imipenemase (IMP)-type metallo-ß-lactamases (MBLs), referred to as IMP-89, IMP-91, and IMP-96, were detected in three clinical isolates from China. Antimicrobial susceptibility tests indicated these novel enzymes were resistant to most ß-lactams, and IMP-96 with a Ser262Gly mutation had higher activity against meropenem than its point mutant. We then collected sequence data on all 91 available IMP variants for phylogenetic analysis. To further analyze the genetic environment of blaIMP, an extensive comparison was applied to nine accessory genetic elements (AGEs), including six sequenced blaIMP-carrying AGEs in this study and three others from GenBank. These nine AGEs were divided into three groups: three IncpJBCL41 plasmids, Tn6417 and its two derivatives, and three Tn6879-related integrative and conjugative elements (ICEs). All blaIMP genes in this study were captured by class 1 integrons. In the integrons, blaIMP genes usually coexisted with other resistance genes, which further impeded clinical antibacterial treatment. The emergence of new IMP variants and the diversity and complexity of their genetic environment make the prevention and control of drug-resistant strains critical, requiring serious attention from clinical and public health management departments. IMPORTANCE The spread of IMP-type MBLs has increased dramatically in recent years. We discovered three novel IMP variants from three clinical isolates in China. We summarized the classification and evolutionary relationship of all available IMP variants. Moreover, we detailed the genetic characteristics of blaIMP-carrying accessory genetic elements in five clinical isolates. Given the risk of rapid and extensive spread of blaIMP genes, we suggest that continuous surveillance is crucial to combat the acquisition and transmission of blaIMP genes by bacteria, which can impede clinical therapy effectiveness.

Comparative evaluation of phenotypic and genotypic methods for the rapid and cost-effective detection of carbapenemases in extensively drug resistant Klebsiella pneumoniae.

PURPOSE: Carbapenemases are the enzymes that can hydrolyze carbapenems and other ß-lactam antibiotics. These enzymes confer resistance to multiple antibiotics and act as a stumbling block in the treatment of infections caused by gram-negative bacteria. Therefore, rapid and specific detection of these enzymes is crucial for deciding the course of treatment and better clinical outcomes. MATERIAL AND METHODS: This study was conducted to compare various phenotypic and PCR based methods for the detection of carbapenemases in carbapenem- and colistin-resistant Klebsiella pneumoniae. One hundred clinical isolates of extensively resistant Klebsiella pneumoniae were included in the study. Phenotypic detection for carbapenemases was performed by Rapidec® Carba NP (Biomerieux), modified carbapenem inactivation method (mCIM), imipenem-ethylenediaminetetraacetic acid disk synergy (EDS), double disk synergy test using mercaptopropionic acid (DDST-MPA), and combined disk method (CD) and for colistin by microbroth dilution method. Genotypic detection for carbapenemases and colistin resistance was performed by targeted PCR. RESULTS: The sensitivity of Carba NP test and mCIM were positive in 95% and 96% respectively and specificity was 100% for both methods. The sensitivity of EDS, DDST-MPA, and CD were 55.6%, 88.9% and 54.5% respectively. Among the carbapenem resistance genes, blaOXA-48 (82%) genes were the most prevalent. Among metallo-beta lactamases, blaVIM (56%) was most common followed by blaNDM (54%) and blaIMP (20%). The mcr-1 gene for colistin resistance was not detected in any isolate. CONCLUSION: Among the five phenotypic assays analyzed, the mCIM is the most simple, inexpensive, accurate and reproducible method for carbapenemase detection in Klebsiella pneumoniae. The DDST-MPA test provides the best sensitivity for the detection of carbapenemases, although specificity is low. These tests, when applied in a clinical laboratory and assessed by the microbiologist, can help in guiding the course of treatment.

Molecular detection of Metallo-Beta-Lactamase and alginate in multidrug resistance Pseudomonas aeruginosa isolated from the clinical specimen.

Pseudomonas aeruginosa pathogen is opportunistic. Several virulence factors and biofilms can cause its pathogenicity. Furthermore, infections triggered via multidrug-resistant P. aeruginosa among hospitalized patients are a public health concern. The primary antimicrobial agents in treating Gram-negative infection include Meropenem and Imipenem. Moreover, the spread of Carbapenem-resistant P. aeruginosa is a focal concern worldwide. The present research aims to determine the spread of Carbapenem-resistant P. aeruginosa, and the distribution of the Alginate and Metallo-beta-lactamase encoding gene in clinical isolates. In the present cross-sectional descriptive research, 50 wound and sputum clinical specimens were obtained. Isolates were all identified by applying cultural characteristics and biochemical tests. The Polymerase Chain Reaction (PCR) was conducted to distinguish algD, BLA-VIM, BLA-IMP, and 16SrRNA genes. Moreover, the phenotypic method was used to detect hemolysin. The disk diffusion technique was applied to screen clinical isolates for eight antimicrobial agents. The PCR results showed all isolates to be positive for algD and negative for BLA-VIM and BLA-IMP genes. Hemolysin and multidrug resistance prevalence was 100% and 76%, respectively. Furthermore, Meropenem proved to be the most efficient antibiotic against clinical isolates. Alginate and hemolysin are considered significant virulence factors for P. aeruginosa, playing a key role in triggering diseases and tissue or skin lesions. The emergence of Multidrug Resistant (MDR) isolates indicates that developing antibiotic stewardship in our regional community hospital is a top priority. Infection control measures could help control the distribution of virulence genes in P. aeruginosa isolates. Moreover, regular observation is needed to decrease public health threats, distributing virulence factors and Imipenem-resistance patterns in clinical isolates of P. aeruginosa.

Development of Peptide-based Metallo-ß-lactamase Inhibitors as a New Strategy to Combat Antimicrobial Resistance: A Mini-review.

Global dissemination of antimicrobial resistance (AMR) not only poses a significant threat to human health, food security, and social development but also results in millions of deaths each year. In Gram-negative bacteria, the primary mechanism of resistance to ß-lactam antibiotics is the production of ß-lactamases, one of which is carbapenem-hydrolyzing ß-lactamases known as carbapenemases. As a general scheme, these enzymes are divided into Ambler class A, B, C, and D based on their protein sequence homology. Class B ß-lactamases are also known as metallo-ß-lactamases (MBLs). The incidence of recovery of bacteria expressing metallo-ß- lactamases (MBLs) has increased dramatically in recent years, almost reaching a pandemic proportion. MBLs can be further divided into three subclasses (B1, B2, and B3) based on the homology of protein sequences as well as the differences in zinc coordination. The development of inhibitors is one effective strategy to suppress the activities of MBLs and restore the activity of ß-lactam antibiotics. Although thousands of MBL inhibitors have been reported, none have been approved for clinical use. This review describes the clinical application potential of peptide-based drugs that exhibit inhibitory activity against MBLs identified in past decades. In this report, peptide-based inhibitors of MBLs are divided into several groups based on the mode of action, highlighting compounds of promising properties that are suitable for further advancement. We discuss how traditional computational tools, such as in silico screening and molecular docking, along with new methods, such as deep learning and machine learning, enable a more accurate and efficient design of peptide-based inhibitors of MBLs.

Recent developments in the medicinal chemistry of single boron atom-containing compounds.

Various boron-containing drugs have been approved for clinical use over the past two decades, and more are currently in clinical trials. The increasing interest in boron-containing compounds is due to their unique binding properties to biological targets; for example, boron substitution can be used to modulate biological activity, pharmacokinetic properties, and drug resistance. In this perspective, we aim to comprehensively review the current status of boron compounds in drug discovery, focusing especially on progress from 2015 to December 2020. We classify these compounds into groups showing anticancer, antibacterial, antiviral, antiparasitic and other activities, and discuss the biological targets associated with each activity, as well as potential future developments.

Antimicrobial Activity of Aztreonam in Combination with Old and New ß-Lactamase Inhibitors against MBL and ESBL Co-Producing Gram-Negative Clinical Isolates: Possible Options for the Treatment of Complicated Infections.

Metallo-ß-lactamases (MBLs) are among the most challenging bacterial enzymes to overcome. Aztreonam (ATM) is the only ß-lactam not hydrolyzed by MBLs but is often inactivated by co-produced extended-spectrum ß-lactamases (ESBL). We assessed the activity of the combination of ATM with old and new ß-lactamases inhibitors (BLIs) against MBL and ESBL co-producing Gram-negative clinical isolates. Six Enterobacterales and three non-fermenting bacilli co-producing MBL and ESBL determinants were selected as difficult-to-treat pathogens. ESBLs and MBLs genes were characterized by PCR and sequencing. The activity of ATM in combination with seven different BLIs (clavulanate, sulbactam, tazobactam, vaborbactam, avibactam, relebactam, zidebactam) was assessed by microdilution assay and time-kill curve. ATM plus avibactam was the most effective combination, able to restore ATM susceptibility in four out of nine tested isolates, reaching in some cases a 128-fold reduction of the MIC of ATM. In addition, relebactam and zidebactam showed to be effective, but with lesser reduction of the MIC of ATM. E. meningoseptica and C. indologenes were not inhibited by any ATM-BLI combination. ATM-BLI combinations demonstrated to be promising against MBL and ESBL co-producers, hence providing multiple options for treatment of related infections. However, no effective combination was found for some non-fermentative bacilli, suggesting the presence of additional resistance mechanisms that complicate the choice of an active therapy.

Co-Existence of Certain ESBLs, MBLs and Plasmid Mediated Quinolone Resistance Genes among MDR E. coli Isolated from Different Clinical Specimens in Egypt.

The emergence of multi-drug resistant (MDR) strains and even pan drug resistant (PDR) strains is alarming. In this study, we studied the resistance pattern of E. coli pathogens recovered from patients with different infections in different hospitals in Minia, Egypt and the co-existence of different resistance determinants. E. coli was the most prevalent among patients suffering from urinary tract infections (62%), while they were the least isolated from eye infections (10%). High prevalence of MDR isolates was found (73%) associated with high ESBLs and MBLs production (89.4% and 64.8%, respectively). blaTEM (80%) and blaNDM (43%) were the most frequent ESBL and MBL, respectively. None of the isolates harbored blaKPC and blaOXA-48 carbapenemase like genes. Also, the fluoroquinolone modifying enzyme gene aac-(6')-Ib-cr was detected in 25.2% of the isolates. More than one gene was found in 81% of the isolates. Azithromycin was one of the most effective antibiotics against MDR E. coli pathogens. The high MAR index of the isolates and the high prevalence of resistance genes, indicates an important public health concern and high-risk communities where antibiotics are abused.

Management of serious infections caused by metallo ß-lactamases with or without OXA-48-like expressing Enterobacterales with aztreonam and ceftazidime/avibactam combination: Dosing strategy for better clinical outcome.

Serious infections caused by MBLs with or without OXA-48-like expressing Enterobacterales remain challenging to treat. Since aztreonam is stable to MBLs, it can be combined with ceftazidime/avibactam to protect against concurrently expressed ESBLs and class C ß-lactamases in MBL pathogens. However, in the light of dose-limiting hepatotoxicity of aztreonam, short half life of avibactam, significant protein binding of aztreonam, appropriate dosing and method of administration to optimize PK/PD and toxicodynamics for this combination is being debated. Based on in-vitro PK/PD studies, simultaneous administration of 6/1.5 g of ceftazidime/avibactam and 8 g of aztreonam per day has been recently suggested.

Discovery of thiosemicarbazone derivatives as effective New Delhi metallo-ß-lactamase-1 (NDM-1) inhibitors against NDM-1 producing clinical isolates.

New Delhi metallo-ß-lactamase-1 (NDM-1) is capable of hydrolyzing nearly all ß-lactam antibiotics, posing an emerging threat to public health. There are currently less effective treatment options for treating NDM-1 positive "superbug", and no promising NDM-1 inhibitors were used in clinical practice. In this study, structure-activity relationship based on thiosemicarbazone derivatives was systematically characterized and their potential activities combined with meropenem (MEM) were evaluated. Compounds 19bg and 19bh exhibited excellent activity against 10 NDM-positive isolate clinical isolates in reversing MEM resistance. Further studies demonstrated compounds 19bg and 19bh were uncompetitive NDM-1 inhibitors with Ki = 0.63 and 0.44 µmol/L, respectively. Molecular docking speculated that compounds 19bg and 19bh were most likely to bind in the allosteric pocket which would affect the catalytic effect of NDM-1 on the substrate meropenem. Toxicity evaluation experiment showed that no hemolysis activities even at concentrations of 1000 mg/mL against red blood cells. In vivo experimental results showed combination of MEM and compound 19bh was markedly effective in treating infections caused by NDM-1 positive strain and prolonging the survival time of sepsis mice. Our finding showed that compound 19bh might be a promising lead in developing new inhibitor to treat NDM-1 producing superbug.

Structure-guided optimization of D-captopril for discovery of potent NDM-1 inhibitors.

ß-lactam antibiotics have long been the mainstay for the treatment of bacterial infections. New Delhi metallo-ß-lactamase 1 (NDM-1) is able to hydrolyze nearly all ß-lactam antibiotics and even clinically used serine-ß-lactamase inhibitors. The wide and rapid spreading of NDM-1 gene among pathogenic bacteria has attracted extensive attention, therefore high potency NDM-1 inhibitors are urgently needed. Here we report a series of structure-guided design of D-captopril derivatives that can inhibit the activity of NDM-1 in vitro and at cellular levels. Structural comparison indicates the mechanisms of inhibition enhancement and provides insights for further inhibitor optimization.

Design, synthesis, biological evaluation and in silico studies of certain aryl sulfonyl hydrazones conjugated with 1,3-diaryl pyrazoles as potent metallo-ß-lactamase inhibitors.

Based on a structure-guided approach, aryl sulfonyl hydrazones conjugated with 1,3-diaryl pyrazoles were designed to target metallo-ß-lactamases (MBLs), using Klebsiella pneumoniaeNDM-1 as a model. The in vitro MBLs inhibition showed remarkable inhibition constant for most of the designed compounds at a low micromolar range (1.5-16.4 µM) against NDM-1, IMP-1 and AIM-1 MBLs. Furthermore, all compounds showed promising antibacterial activity against (K+, K1-K9) resistant clinical isolates of K. pneumoniae and were able to re-sensitize resistant K. pneumoniae (K5) strain towards meropenem and cefalexin. Besides, in vivo toxicity testing exhibited that the most active compound was non-toxic and well tolerated by the experimental animals orally up to 350 mg/kg and up to 125 mg/kg parenterally. The docking experiments on NDM-1 and IMP-1 rationalized the observed in vitro MBLs inhibition activity. Generally, this work presents a fruitful matrix to extend the chemical space for MBLs inhibition. This aids in tackling drug-resistance issues in antibacterial treatment.

Molecular Epidemiology of Carbapenemase-Producing Pseudomonas aeruginosa Isolated from an Iranian University Hospital: Evidence for Spread of High-Risk Clones.

PURPOSE: Given the importance of treatment failure due to multidrug-resistant (MDR) strains, studies on population structure of these organisms are necessary to improve control strategies. Accordingly, the current study aimed to determine the prevalence of carbapenem-resistant P. aeruginosa (CRPA) at a teaching referral hospital in Iran and to analyz their molecular clonality by multilocus sequence typing (MLST) and pulsed-field gel electrophoresis (PFGE) for epidemiological purposes. METHODS: In this study, modified Hodge test (MHT) and double-disk synergy test (DDST) were used for carbapenemase production and metallo-ß-lactamases (MBLs) screening, respectively. All P. aeruginosa isolates were tested for antimicrobial resistance. Moreover, MBL genes (blaIMP, blaVIM, blaSPM, blaNDM) were detected by multiplex PCR assay. RESULTS: Among 68 P. aeruginosa clinical isolates, 38 (55.88%) isolates were CRPA. Antibiotic susceptibility testing revealed that most of these isolates were MDR. PFGE analyses showed 5 common types and 27 single types among CRPA isolates. MLST analysis revealed three major clusters (MLST-sequence types (STs): 235, 357, and 861) among them. The 30 non-CRPA isolates corresponded mainly to MLST-STs 253, 360, and 446. CONCLUSION: Our results showed that internationally distributed MLST-STs with widely genomic diversity have spread in our hospital, and clonal expansion of MDR strains of P. aeruginosa was described as well.

Prevalence and Mechanisms of Carbapenem Resistance Among Acinetobacter baumannii Clinical Isolates in Egypt.

The increasing number of carbapenem-resistant Acinetobacter baumannii clinical isolates is a major concern, which restricts therapeutic options for treatment of serious infections caused by this emerging pathogen. The aim of this work is to assess the antimicrobial resistance profile and identify the molecular mechanisms involved in carbapenem resistance in A. baumannii isolated from different clinical sources in Mansoura University Hospitals, Egypt. Antimicrobial susceptibility testing has shown that resistance to carbapenem has dramatically increased (98%) with concomitant elevated levels of resistance to quinolones, trimethoprim/sulfamethoxazole, and aminoglycosides. Polymyxin B and colistin are considered the last resort. Random amplified polymorphic DNA (RAPD) typing method revealed great diversity among A. baumannii isolates. Coexistence of diverse intrinsic and acquired carbapenem-hydrolyzing ß-lactamases has been detected in the tested isolates: Ambler class A: blaKPC (56%) and blaGES (48%), and Ambler class B: blaNDM (30%), blaSIM (28%), blaVIM (20%), and blaIMP (10%). Most isolates (94%) carried blaOXA-23-like and blaOXA-51-like simultaneously. blaOXA-23-like was preceded by ISAba1 providing a potent promoter activity for its expression. Sequencing analysis revealed that ISAba1 has been also inserted in carbapenem resistance-associated outer membrane protein (OMP) (carO) gene in three isolates, two of which were clonal based on RAPD typing, leading to interruption of its expression as confirmed by SDS-PAGE analysis of OMP fractions. Carbapenem resistance genes are widely distributed among A. baumannii clinical isolates from different clinical sources. Therefore, enhanced infection control measures, effective barriers, and rational use of antimicrobials should be enforced in hospitals for minimizing the widespread resistance to carbapenems and all other antibiotics.

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.

First Report of Klebsiella oxytoca Strain Simultaneously Producing NDM-1, IMP-4, and KPC-2 Carbapenemases.

The nucleotide sequences of five plasmids from one Klebsiella oxytoca isolate were determined using the PacBio RS II system. Plasmid analysis revealed that blaNDM-1 was carried on an IncX3 plasmid. The blaIMP-4 and blaKPC-2 genes were located on IncN and IncP-6 plasmids, respectively. Comparative sequence analysis highlighted the successful spread of carbapenemase-harboring plasmids among different enterobacterial species. We report for the first time, to our knowledge, coproducing NDM-1, KPC-2, and IMP-4 carbapenemases on a K. oxytoca isolate.

Carbapenem Susceptibility and Multidrug-Resistance in Pseudomonas aeruginosa Isolates in Egypt.

BACKGROUND: Resistant Pseudomonas aeruginosa is a serious concern for antimicrobial therapy, as the common isolates exhibit variable grades of resistance, involving beta-lactamase enzymes, beside native defense mechanisms. OBJECTIVES: The present study was designed to determine the occurrence of Metallo-ß- Lactamases (MBL) and Amp C harboring P. aeruginosa isolates from Suez Canal university hospital in Ismailia, Egypt. METHODS: A total of 147 P. aeruginosa isolates, recovered from 311 patients during a 10-month period, were collected between May 2013 and February 2014; the isolates were collected from urine, wound and sputum. Minimum inhibitory concentration (MIC) determined by agar dilution methods was ≥2 µg/mL for meropenem and imipenem. Identification of P. aeruginosa was confirmed using API 20NE. Metallo-ß- Lactamases and Amp C were detected based on different phenotypic methods. RESULTS: Overall, 26.5% of P. aeruginosa isolates (39/147) were carbapenem resistant isolates. Furthermore, 64.1% (25/39) were MBL producers, these isolates were screened by the combined disc and disc diffusion methods to determine the ability of MBL production. Both MBL and Amp C harbored P. aeruginosa isolates were 28% (7/25). Sixty-four percent of P. aeruginosa isolates were multidrug resistant (MDR) (16/25). The sensitivity toward polymyxin, imipenem, norfloxacin, piperacillin-tazobactam and gentamicin was 99%, 91%, 88%, 82% and 78%, respectively. The resistance rate towards cefotaxime, ceftazidime, cefepime, aztreonam and meropenem was 98.6%, 86%, 71.4%, 34% and 30%, respectively. CONCLUSIONS: Multidrug resistance was significantly associated with MBL production in P. aeruginosa. Early detection of MBL-producing P. aeruginosa and hospital antibiotic policy prescription helps proper antimicrobial therapy and avoidance of dissemination of these multidrug resistance isolates.

Actividad comparativa in vitro de doripenem y otros carbapenemicos frente a pseudomonas aeruginosa aisladas en el laboratorio de microbiología del hospital Vargas de Caracas / Comparative in vitro activity of dorypenem and other carbapenems in pseudomonas aeruginosa isolates on Vargas Caracas Hospital´s Microbiology

Los ensayos de cuantificación de ARN plasmáticos de VIH-1 son importantes para el control de pacientes infectados, así como el monitoreo de la respuesta a la terapia antirretroviral. Por lo tanto, los ensayos comerciales empleados para este propósito deben presentar buena correlación entre si, para dar lugar al manejo terapéutico apropiado. El objetivo del estudio consistió en correlacionar los resultados obtenidos mediante el ensayo de amplificación de señal (bDNA) y PCR en tiempo real (RT-PCR), ambas casas comerciales aprobadas por la FDA y con diferente diana de detección del VIH-1. La validación se realizó con 180 muestras clínicas de pacientes referidos al INHRR. Los resultados fueron comparados con la subpoblación de linfocitos TCD4+ determinados mediante citometría de flujo. El análisis estadístico se realizó empleando el coeficiente de regresión lineal de Pearson (R2) y el valor de contraste de hipótesis con una significancia del 95 %, usando el programa SPSS Statistics v10.0. Se observó una buena correlación entre los ensayos (R2=0.961, p<0.05), siendo la RTPCR más sensible. Las diferencias cuantitativas de carga viral entre las técnicas ensayadas fue menor de 0.5 log10 copias/ml para el 89% de las muestras, y >1 log10 copias/ml solo en dos pacientes, no indicando necesariamente cambio terapéutico. Adicionalmente, se encontró una correlación inversa entre los linfocitos TCD4+ y carga viral del VIH-1 medida por bDNA (R2= 0.20, p<0.05) y RT-PCR (R2= 0.15, p<0.05). Los ensayos evaluados mostraron que ambas técnicas puedes ser empleadas indistintamente para el control de los pacientes VIH positivo.

The assay for quantification of plasma HIV-1 RNA are important for the control of patients infected, as well as the monitoring of the response to antiretroviral therapy. Therefore, the commercial assays used for this purpose must submit good correlation between to give place to the appropriate therapeutic management. In this study, we correlate the results obtained through the testing of signal amplification (bDNA) and real-time PCR (RT-PCR), two comercial technical approved by the FDA and with different targets of detection HIV-1. The validation was carried out with 180 clinical samples of patients referred to the INHRR. The results were compared with the subpopulation of lymphocytes TCD4+ determined by flow cytometry. The statistical analysis was performed using the program SPSS Statistics v10. It was observed good correlation between the tests studied (R2=0.961, p<0.05), with RT-PCR more sensitive. The quantitative differences in viral load between the techniques tested was less than 0.5 log10 copies/ml for the 89% of the samples, and >1 log10 copies/ml in only two patients. Additionally, it was found an inverse correlation between lymphocytes TCD4+ and viral load of HIV-1, measured by bDNA (R2= 0.20, p<0.05) and RT-PCR (R2= 0.15, p<0.05). Therefore, these assays can be employed for the patient control HIV.

Description of IMP-31, a novel metallo-ß-lactamase found in an ST235 Pseudomonas aeruginosa strain in Western Germany.

OBJECTIVES: The objective of this study was to characterize a novel IMP-type metallo-ß-lactamase (MBL) found in an MDR clinical isolate of Pseudomonas aeruginosa. METHODS: The P. aeruginosa isolate NRZ-00156 was recovered from an inguinal swab from a patient hospitalized in Western Germany and showed high MICs of carbapenems. MBL production was analysed by Etest for MBLs, an EDTA combined disc test and an EDTA bioassay. Typing of the isolate was performed by MLST. Genetic characterization of the new blaIMP gene was performed by sequencing the PCR products. A phylogenetic tree was constructed. The novel blaIMP gene was expressed in Escherichia coli TOP10 and the enzyme was subjected to biochemical characterization. RESULTS: The P. aeruginosa isolate NRZ-00156 expressed the ST235 allelic profile and was resistant to all the ß-lactams tested except aztreonam. The isolate was positive for MBL production and harboured a new IMP allele, blaIMP-31, located on a disrupted class I integron [also carrying the blaOXA-35, aac(6')-Ib, aac(3)-Ic and aphA15 genes]. Its closest relative was IMP-35, with 96.7% amino acid identity. Expression of blaIMP-31 demonstrated that E. coli TOP10 producing IMP-31 had elevated resistance to all the ß-lactams tested except aztreonam. Kinetic data were obtained for both IMP-31 and IMP-1. In comparison with IMP-1, IMP-31 showed weaker hydrolytic activity against all the ß-lactams tested, which resulted from lower kcat values. CONCLUSIONS: The characterization of the new IMP-type gene blaIMP-31 from an ST235 P. aeruginosa isolate indicates an ongoing spread of highly divergent IMP-type carbapenemases in clinical P. aeruginosa strains and highlights the continuous need for the prevention of nosocomial infections caused by MDR Gram-negative bacteria.