Antimicrobial activity evaluation and comparison of methods of susceptibility for Klebsiella pneumoniae carbapenemase (KPC)-producing Enterobacter spp. isolates

Abstract The production of KPC (Klebsiella pneumoniae carbapenemase) is the major mechanism of resistance to carbapenem agents in enterobacterias. In this context, forty KPC-producing Enterobacter spp. clinical isolates were studied. It was evaluated the activity of antimicrobial agents: polymyxin B, tigecycline, ertapenem, imipenem and meropenem, and was performed a comparison of the methodologies used to determine the susceptibility: broth microdilution, Etest® (bioMérieux), Vitek 2® automated system (bioMérieux) and disc diffusion. It was calculated the minimum inhibitory concentration (MIC) for each antimicrobial and polymyxin B showed the lowest concentrations for broth microdilution. Errors also were calculated among the techniques, tigecycline and ertapenem were the antibiotics with the largest and the lower number of discrepancies, respectively. Moreover, Vitek 2® automated system was the method most similar compared to the broth microdilution. Therefore, is important to evaluate the performance of new methods in comparison to the reference method, broth microdilution.

Molecular identification of multidrug resistant Enterobacter hormaechei in Venezuela / Identificación molecular de Enterobacter hormaechei multidrogoresistente en Venezuela

Besides the importance of Enterobacter cloacae species complex as a nosocomial pathogen, little is known about the frequency of each species/genotype. Here, we describe a strain of E. hormaechei subsp. hormaechei isolated from a bronchial secretion of a patient, in the Intensive Care Unit at the General Hospital of Cumaná, Venezuela, who died due to complications of his infection. The molecular identification was done by sequencing the 16S rRNA gene and comparing it to sequences from the GenBank. This strain showed resistance to multiple families of antibiotics (MDR), and the genes blaKPC and blaVIM were detected by PCR. This is the first time E. hormaechei has been identified in Venezuela.

A pesar de la importancia de las especies del complejo Enterobacter cloacae como patógeno nosocomial, poco se conoce sobre la frecuencia de cada especie/genotipo. Aquí se describe una cepa de E. hormaechei subsp. hormaechei aislada de una secreción bronquial de un paciente internado en la Unidad de Cuidados Intensivos del Hospital General de Cumaná, Venezuela, quien murió producto de complicaciones de su infección. La identificación molecular fue hecha por secuenciación del gen ARNr 16S y porcomparación con las secuencias del GenBank. Esta cepa mostró resistencia a múltiples familias de antibióticos (MDR) y se detectaron los genes blaKPCyblaVIMpor PCR. Este es el primer reporte de E. hormaechei en Venezuela.

Antimicrobial Effect and Surface Tension of Some Chelating Solutions with Added Surfactants

Abstract This study assessed the antimicrobial efficacy and surface tension of established irrigating solutions with a new experimental chelating solution in infected dentin tubes. Twenty-five specimens were randomly assigned to each of the irrigating solutions. Twenty specimens were used as negative and positive controls. After 21 days of contamination with E. faecalis, the irrigating solutions MTAD, QMiX and Tetraclean NA were delivered into each infected root canal. The solutions were removed and dentin samples were withdrawn from the root canals with sterile low-speed round burs with increasing ISO diameters. The dentin powder samples obtained with each bur were immediately collected in separate test tubes containing 3 mL of BHI broth. After that, 100 μL from each test tube was cultured on blood agar. The grown colonies were counted and recorded as colony-forming units (CFU). The surface tension of the irrigants was measured using a Cahn DCA-322 Dynamic Contact Angle Analyzer. A Kruskal Wallis nonparametric ANOVA and a Friedman test were used (p<0.05). Tetraclean NA showed lower surface tension and CFU values than MTAD and QMiX. Better antibacterial action and low surface tension were observed for Tetraclean NA, probably due to the improved penetration into the root canal and dentinal tubes

Resumo Este estudo avaliou a eficácia antimicrobiana e tensão superficial de soluções irrigadoras e uma nova solução quelante em tubos de dentina infectada. Vinte e cinco espécimes foram aleatoriamente distribuídos conforme as soluções irrigantes. Decorrifdos 21 dias de contaminação com E. faecalis, a soluções de irrigação MTAD, QMiX e Tetraclean NA foram distribuídas em cada canal radicular infectado. As soluções foram removidas e as amostras de dentina foram retiradas dos canais radiculares com brocas esféricas de baixa velocidade com diâmetros ISO sucessivamente maiores. As amostras do pó de dentina obtidas com cada broca foram imediatamente colocadas em tubos de ensaio separados contendo 3 mL de caldo BHI. A seguir, 100 μL de cada amostra do tubo de teste foi cultivada em agar de sangue. As colônias crescidas foram contadas e registadas como unidades formadoras de colônias (UFC). A tensão superficial das soluções irrigantes foi medida utilizando o método de Wilhelmy. A análise não paramétrica de Kruskal-Wallis e o teste de Friedman foram utilizados (p<0,05). Tetraclean NA apresentou menor tensão de superfície e menores valores de UFC do que MTAD e QMiX. A melhor ação antibacteriana e baixa tensão superficial foram observadas para Tetraclean NA, provavelmente devido à melhor penetração no canal radicular e túbulos dentinários.

Plant Growth Promotion and Root Colonization by EPS Producing Enterobacter sp. RZS5 under Heavy Metal Contaminated Soil.

The heavy metal resistant bacterium isolated from field soil and identified as Enterobacter sp. RZS5 tolerates a high concentration (100-2000 mM) of various heavy metal ions such as Mn2+, Ni2+, Zn2+, Cu2+, CO2+ and Fe2+ when grown in such environment and produces exopolysaccharides (EPS). Here, we have demonstrated EPS production by Enterobacter sp. RZS5 during 60 h of growth in yeast extract mannitol broth (YEMB). The yield increased by two fold after the addition of 60 M of Ca2+; 50 M of Fe2+ and 60 M of Mg2+ ions in YEMB, and the optimization of physico-chemical parameters. EPS was extracted with 30% (v/v) of isopropanol as against the commonly used 50% (v/v) isopropanol method. EPS-rich broth promoted seed germination, shoot height, root length, number of leaves and chlorophyll content of wheat (Triticum aestivum) and peanut (Arachis hypogaea) seeds. The higher colony-forming unit of Enterobacter sp. in soil inoculated with EPS rich broth of Enterobacter sp. indicated the root colonizing potential and rhizosphere competence of the isolate. The FTIR spectra of the EPS extract confirmed the presence of the functional group characteristics of EPS known to exhibit a high binding affinity towards certain metal ions. This overall growth and vigour in plants along with the effective root colonization, reflected the potential of the isolate as an efficient bio-inoculant in bioremediation.

Isolation and characterization of arsenic resistant bacteria from wastewater

The present study proposed the isolation of arsenic resistant bacteria from wastewater. Only three bacterial isolates (MNZ1, MNZ4 and MNZ6) were able to grow in high concentrations of arsenic. The minimum inhibitory concentrations of arsenic against MNZ1, MNZ4 and MNZ6 were 300 mg/L, 300 mg/L and 370 mg/L respectively. The isolated strains showed maximum growth at 37 ºC and at 7.0 pH in control but in arsenite stress Luria Bertani broth the bacterial growth is lower than control. All strains were arsenite oxidizing. All strains were biochemically characterized and ribotyping (16S rRNA) was done for the purpose of identification which confirmed that MNZ1 was homologous to Enterobacter sp. while MNZ4 and MNZ6 showed their maximum homology with Klebsiella pneumoniae. The protein profiling of these strains showed in arsenic stressed and non stressed conditions, so no bands of induced proteins appeared in stressed conditions. The bacterial isolates can be exploited for bioremediation of arsenic containing wastes, since they seem to have the potential to oxidize the arsenite (more toxic) into arsenate (less toxic) form.

Effects of different osmolarities on bacterial biofilm formation

Biofilm formation depends on several factors. The influence of different osmolarities on bacterial biofilm formation was studied. Two strains (Enterobacter sp. and Stenotrophomonas sp.) exhibited the most remarkable alterations. Biofilm formation is an important trait and its use has been associated to the protection of organisms against environmental stresses.

FIRST REPORT OF METALLO-β-LACTAMASES PRODUCING Enterobacter spp. STRAINS FROM VENEZUELA / Primer reporte de cepas de Enterobacter spp productoras de metalobetalactamasas de Venezuela

Clinical strains of Enterobacter were isolated from Cumana's Central Hospital in Venezuela, and classified as E. cloacae (21), E. aerogenes (7), E. intermedium (1), E. sakazakii (1) and three unclassified. The strains showed high levels of resistance, especially to SXT (58.1%), CRO (48.8%), CAZ (46.6%), PIP (46.4%), CIP (45.2%) and ATM (43.3%). This is the first report for South America of blaVIM-2 in two E. cloacae and one Enterobacter sp., which also showed multiple mechanisms of resistance. Both E. cloacae showed blaTEM-1, but only one showed blaCTX-M-15 gene, while no blaSHV was detected.

Cepas clínicas de Enterobacter fueron aisladas del Hospital central de Cumaná en Venezuela, y se clasificaron como E. cloacae (21), E. aerogenes (7), E. intermedium (1), E. sakazakii (1) y 3 sin clasificar. Las cepas mostraron altos niveles de resistencia, especialmente a SXT (58.1%), CRO (48.8%), CAZ (46.6%), PIP (46.4%), CIP (45.2%) and ATM (43.3%). Este es el primer reporte de América del Sur de blaVIM-2 en dos cepas de E. cloacae y una de Enterobacter sp., las cuales también mostraron múltiples mecanismos de resistencia. Ambas especies de E. cloacae mostraron genes blaTEM-1, pero solo una mostro el gen blaCTX-M-15, mientras que blaSHV no fue detectado.

Genes blaTEM, blaSHV y blaCTX-M en enterobacterias productoras de b-lactamasas de espectro extendido aisladas de pacientes con infección intrahospitalaria / blaTEM, blaSHV y blaCTX-M genes in extended-spectrum b-lactamases producing enterobacterias isolated from patients with hospital-acquired infections

El objetivo de este estudio fue identificar los genes blaTEM, blaSHV y blaCTX-M en aislados clínicos de enterobacterias productoras de b-lactamasas de espectro extendido (BLEE), recolectadas entre septiembre y noviembre de 2005. Además de la resistencia a las cefalosporinas de tercera generación, los aislados también mostraron resistencia a cloranfenicol (59,2%) amikacina (37,0%) y gentamicina (40,7%) y se mostraron sensibles a imipenem y meropenem. Nueve cepas lograron transferir la resistencia a las cefalosporinas de tercera generación, así como la producción de BLEE. En los aislados clínicos se detectaron los genes blaSHV, blaTEM y blaCTX-M, donde los tipos blaTEM-1, blaSHV-1, blaSHV-5 blaSHV-5-2a y blaCTX-M-1 fueron los prevalentes; mientras que en las transconjugantes sólo se detectaron blaTEM-1, blaSHV-5 y blaSHV-5-2a. Se identificaron en total siete tipos de genes, de los cuales cinco eran codificantes de enzimas tipo BLEE, lo que demuestra que en el centro hospitalario la resistencia a las cefalosporinas de tercera generación es debida a diversas enzimas.

The objective of the present investigation was to identify the blaTEM, blaSHV and blaCTX-M genes on extended-spectrum b-lactamases (ESBL) producing Enterobacteriaceae from clinical isolates, collected between September and November 2005. In addition to third-generation cephalosporin resistance, the isolates also showed resistance to chloramphenicol (59.2%), amikacin (37.0%) and gentamicin (40.7%), and demonstrated sensitivity to imipenem and meropenem. Nine strains were capable of transferring third-generation cephalosporin resistance, as well as the production of ESBL. In the clinical isolates, the genes blaSHV, blaTEM and blaCTX-M were detected, being more prevalent the types blaTEM-1, blaSHV-1, blaSHV-5 blaSHV-5-2a and blaCTX-M-1; while in the trans-conjugated only blaTEM-1, blaSHV-5 y blaSHV-5-2a were found. In total, seven types of genes were identified, five of which were codifying genes for ESBL-type enzymes. This demonstrates that in the hospital center, resistance to third-generation cephalosporin is mediated by several enzymes.

Phenotypic & molecular characterization of AmpC β-lactamases among Escherichia coli, Klebsiella spp. & Enterobacter spp. from five Indian Medical Centers.

Background & objectives: AmpC β-lactamases which are often plasmid mediated hydrolyze all β-lactam antibiotics except cefepime and carbapenems. We evaluated the presence of AmpC β-lactamases among Enterobacteriaceae strains recovered prospectively from patients at five Indian tertiary care centres. Methods: The study included 909 consecutive Gram-negative isolates recovered from clinically significant specimens during June 2007 - May 2008 as part of an ICMR-ESBL study. Among the study isolates, 312 were found to be cefoxitin resistant by disc diffusion test (DDT). Minimum inhibitory concentration (MIC) determination by E test was done against amikacin, levofloxacin, impinem, meropenem, ertapenem, tigecycline and piperacillin-tazobactam. Combined DDT using phenyl boronic acid as inhibitor with cefoxitin was used for phenotypic confirmation of AmpC phenotype. The common Amp C genotypes ACC, FOX, MOX, DHA, CIT and EBC were detected by multiplex PCR. Results: Plasmid mediated Amp C phenotype was confirmed in 114 of the 312 (36.5%) cefoxitin resistant isolates with 255 (81.7%) showing multidrug resistance. Susceptibility to tigecycline was highest (99%) followed by imipenem, meropenem (97%), ertapenem (89%), amikacin (85%), and piperacillin-tazobactam (74.6%). Levofloxacin resistance was 82 per cent. ESBL co carriage was observed among 92 per cent of Amp C producers. Among 114 Amp C producers, 48 could be assigned a genotype, this included CIT- FOX (n=25), EBC (n=10), FOX (n = 4), CIT (n=3), EBC-ACC (n=2) and one each of DHA, EBC-DHA, FOX -DHA and FOX-EBC-DHA. Interpretation & Conclusions: Overall, AmpC phenotypes were found in 12.5 per cent isolates, multidrug resistance and ESBL co-carriage among them was high suggesting plasmid mediated spread. The study results have implications in rational antimicrobial therapy and continued surveillance of mechanisms of resistance among nosocomial pathogens.

Increasing antibiotic resistance among uropathogens isolated during years 2006-2009: impact on the empirical management

Urinary tract infections (UTI) are one of the most common infections with an increasing resistance to antimicrobial agents. PURPOSE: Empirical initial antibiotic treatment of UTI must rely on susceptible data from local studies. MATERIALS AND METHODS: Retrospective analysis of isolated bacteria from children with UTIs was performed at the university hospital during years 2006-2009. The findings were compared with data collected in a similar study carried out in 2002- 2003. RESULTS: A total of 1439 uropathogens were isolated. Escherichia coli (E.coli) was the leading cause, followed by Enterobacter, and other gram negative bacilli. It was observed resistance of E.coli to ceftriaxone, cefexime, amikacin, gentamycin, and nalidixic acid; Enterobacter to cefexime; and the resistance of gram negative bacilli to gentamicin and cefexime increased significantly. The highest effective antibiotic was Imipenem, ciprofloxacin, and amikacin with 96.7%, 95% and 91% sensitivity rates , respectively, followed by ceftriaxone 77.2%, gentamicin 77%, nitrofurantoin 76.4%, nalidixic acid 74.3% and cefexime with 70%. CONCLUSION: The use of nitrofurantoin or nalidixic acid as initial empirical antibacterial therapy for cystitis seems appropriate. For cases of simple febrile UTI, the use of initial parenteral therapies with amikacin or ceftriaxone followed by an oral third generation cephalosporin also seemed appropriated, and in cases of severely ill patients or complicated UTI, imipenem as monotherapy or, a combination of Ceftriaxone with an aminoglycoside, are recommended.

Growth response of maize plantlets inoculated with Enterobacter spp., as a model for alternative agriculture / Respuesta de plántulas de maíz inoculadas con Enterobacter spp. como un modelo de agricultura alternativa

A maize rhizosphere isolate was phenotypically and genotypically characterized and identifed as Enterobacter spp. bacterium. Germinated seeds were inoculated, the plantlets were sown in vermiculite and in soil and grown under laboratory and feld conditions, respectively. The adherence, colonization and plant growth promotion capability of Enterobacter sp. UAPS03001 was evaluated in "Rojo-Criollo" maize under laboratory conditions. Twenty days after inoculation, the treated plantlets showed larger biomass than non-inoculated ones. In feld grown plants, the kernel biomass was also greater in inoculated than in non-inoculated plants. The inoculation of maize sprouts with plant growth- promoting bacteria before their sowing in the feld would be an alternative practice for achieving successful yield in temporal agriculture.

En este trabajo se aisló una bacteria de la rizósfera de maíz, que fue caracterizada mediante métodos fenotípicos y genotípicos e identifcada como Enterobacter sp. UAPS03001. La bacteria fue inoculada en semillas de maíz "Rojo-Criollo" germinadas en forma axénica. Las semillas germinadas e inoculadas se plantaron en vermiculita y posteriormente las plántulas fueron cultivadas en vermiculita o en suelo, para evaluar el efecto promotor del crecimiento vegetal de dicha bacteria, bajo condiciones de laboratorio y de campo. Bajo condiciones de laboratorio, también se evaluó la capacidad de esta cepa para adherirse a las plantas de maíz y colonizarlas. Veinte días después de la inoculación, las plántulas inoculadas mostraron una biomasa mayor con referencia a las no inoculadas. En campo, la biomasa de la mazorca fue también mayor en las plantas inoculadas respecto de las plantas no inoculadas. La inoculación de germinados de maíz con una bacteria promotora del crecimiento vegetal y su posterior transferencia a campo podría ser una práctica alternativa para llevar a cabo una producción exitosa en agricultura de temporal.

Sequence analysis of bla CTX-M-28 , an ESBL responsible for third-generation cephalosporin resistance in Enterobacteriaceae, for the first time in India.

The most common group of ESBLs not belonging to the bla TEM or bla SHV families were termed bla CTX-M , to highlight their ESBLs' greater activity against cefotaxime than against ceftazidime. The presence of nosocomial bla CTX-M-28 -producing Enterobacteriaceae strains has not been reported earlier in Indian hospitals. The sequences of bla CTX-M-28 gene from cephalosporin-resistant Enterobacteriaceae were analyzed. The structural gene encodes a 290 amino-acid protein, which is most related to the bla CTX-M beta-lactamases. The conserved K-T-G was identified in the bla CTX-M-28 protein sequence, but significantly, two point mutations (N-->T) and (F-->S) were identified in the Y-G-N- and S-T-F-K-conserved motifs respectively. These point mutations were seen in all the three sequenced isolates.

The prevalence of extended-spectrum beta-lactamase in environmental isolates of Enterobacter.

The incidence of extended-spectrum beta-lactamase (ESBL)-producing strains and multidrug-resistant strains of Enterobacter spp. isolated from the 1312 km long river Narmada was investigated. Out of the 57 isolates of Enterobacter, 73.68% were found to be ESBL producers including the isolates of E. taylorae and isolates of E. agglomerans, which have been characterized for the first time. All the isolates were found susceptible to the antibiotic imipenem. AmpC gene was found in all the Enterobacter strains tested. AmpC beta-lactamase-producing bacterial pathogens may cause major therapeutic failure if not detected and reported in time. It was seen that these enzymes are mainly chromosomally mediated along with several non-AmpC beta-lactamase.

[Antimicrobial susceptibilities of emterobacteriaceae isolated from urinary tract infections in the area of Monastir]

The aim of this study is to determinate the local epidemiology of urinary tract infections in the university hospital of Monastir and to evaluate antimicrobial susceptibility of most incriminated Enterobacteriaceae strains since 2002 to 2005. Identification of strains was based on conventional bacteriological features. Susceptibility to antibiotics was studied according to the Antibiogram Committee of the French Microbiology Society recommendations. In total, 8505 Enterobacteriaceae strains were isolated including Escherichia coli [76%] followed by Klebsiella spp. [10.5%] and Proteus mirabilis [4%]. For E. coli and P. mirabilis, resistances rates were respectively 61% and 71% to amoxicillin, 46.4% and 45.5% to the combination amoxicillin-clavulanic acid, and 39.6% and 26% to cotrimoxazole. Strains of Klebsiella spp. were resistant to cefotaxim and amoxicillin + clavulanic acid in respectively 42% and 20% of cases. Finally, from 2002 to 2005, a significant increase in Enterobacteriaceae resistance was observed for ofloxacin, from 11.9% to 17.6% and ciprofloxacin, from 9.1% to 14.5%. The emergence of antimicrobial resistance justifies that empiric treatment of urinary tract infections should be revised regularly

Antibiotic resistance pattern gramong gram-negative bacteria isolated from raw milk in multan

The use of raw milk in Pakistan has led to the antibiotics resistance. This investigation has showed incidence and antibiotic resistance among Coliform and Klebsiella to nalidixic acid, urixin and little to chloramphenicol. Enterobacter was sensitive to nalidixic acid, tetracycline and showed little resistance to chloramphenicol. E. coli were very sensitive to nalidixic acid, tetracycline, chloramphenicol, urixin, amoxil and with no resistance to ceftriaxone

Invitro comparative evaluation of aminoglycosides at a cancer centre.

A comparative study of in vitro activity of amikacin, gentamicin, netilmicin, tobramycin and isepamicin was done. A total of 200 clinical isolates of Gram negative organisms from various clinical sources were tested. E.Coli was the most frequently isolated organism followed by Pseudomonas spp., Klebsiella spp., Proteus spp., Acinetobacter spp. and Enterobacter spp. Of the 5 aminoglycosides tested, isepamicin showed the highest numbers of susceptible isolates followed by amikacin, netilmicin, gentamicin and tobramycin. MIC 90 value of isepamicin was lower as compared to amikacin.

Mechanisms of bacterial resistance to antimicrobial agents

The 20th century has been considered the antimicrobial era - whereas the 21st century may well represent the post-antimicrobial era. The reason for this dramatic change, should it come to pass, is the development of bacterial resistance to antimicrobial agents. This emerging resistance is now challenging the clinical utility of many antimicrobial agents such that the chemotherapy of hospitalized patients with serious infections has been compromised. If the problem with resistance is to be successfully dealt with by clinicians, the mechanisms of such resistance must be known and understood. This paper thus reviews the most important mechanisms of resistance as well as some of the most important pathogens having these mechanisms. An understanding of these important microbial resistance mechanisms will help the clinician identify circumstances in which resistance may be a problem as well as evaluating the potential usefulness of an alternate antimicrobial agent against resistant microbes

Results of the 1997 SENTRY Antimicrobial Surveillance Program in three Brazilian medical centers

The SENTRY Antimicrobial Surveillance Program began in January, 1997, and is designed to monitor nosocomial and selected community acquired infections via a worldwide surveillance network of sentinel hospitals distributed equally by geographic location and size. Three sites in Brazil - Rio de Janeiro, Florianópolis, and Säo Paulo - participated in the SENTRY Antimicrobial Surveillance Program stet. Rank order of occurrence and antimicrobial susceptibillity of pathogenic species causing bloodstream infections, pneumonia, wound or skin and soft tissue infections, and urinary tract infections (UTI) in hospitalized patients were determined by collecting consecutive isolates over a specified period of time. Antimicrobial susceptibilities of Streptococcus pneumoniae, Haemophilus influenzae, and Moraxella catarrhalis obtained from outpatients with respiratory tract infections were also evaluated. The isolates for the evaluated infections were: 1) bloodstream - 20 consecutive isolates in each calendar month during a 12-month period; 2) pneumonia - 100 consecutive isolates over a 6 month period; 3) wound or skin and soft tissue - 50 consecutive isolates over a 3 month period; and 4) UTI - 50 consecutive isolates over a 3 month period. Each hospital also contributed, over a 6 month period, consecutive clinically significant outpatient isolates (one isolate per patient) of S. pneumoniae, H. influenzae, and M. catarrhalis that were considered pathogens in respiratory tract infections. Data collected for each isolate included species identification, antimicrobial susceptibility profile, data of isolation, and specimen type. Molecular studies were performed on selected isolates. A total of 1,241 bacterial strains were obtained; the majority were cultured from hospitalized patients, while 139 were fastidious organisms from community acquired respiratory tract infections. Gram-negative bacilli and S. aureus were the predominant pathogens, and Enterobacter spp. was a significant pathogen. The predominance of P. aeruginosa and Acinetobacter spp. and the significant levels of resistence to most agents are of major concern, as is the epidemic rate of ESBL-producing strains of Klebsiella spp. and E. coli in Brazil, which is much higher than rates seen in other areas of the world. Resistance among P. aeruginosa and the Enterobacteriaceae to fluoroquinolones, oxacillin-resistant S. aureus, and penicillin- and trimethoprim-sulfamethoxazole-resistant pneumococci were other...