Evidences of gentamicin resistance amplification in Klebsiella pneumoniae isolated from faeces of hospitalized newborns

The intestinal microbiota, a barrier to the establishment of pathogenic bacteria, is also an important reservoir of opportunistic pathogens. It plays a key role in the process of resistance-genes dissemination, commonly carried by specialized genetic elements, like plasmids, phages, and conjugative transposons. We obtained from strains of enterobacteria, isolated from faeces of newborns in a university hospital nursery, indication of phenothypical gentamicin resistance amplification (frequencies of 10-3 to 10-5, compatible with transposition frequencies). Southern blotting assays showed strong hybridization signals for both plasmidial and chromossomal regions in DNA extracted from variants selected at high gentamicin concentrations, using as a probe a labeled cloned insert containing aminoglycoside modifying enzyme (AME) gene sequence originated from a plasmid of a Klebsiella pneumoniae strain previously isolated in the same hospital. Further, we found indications of inactivation to other resistance genes in variants selected under similar conditions, as well as, indications of co-amplification of other AME markers (amikacin). Since the intestinal environment is a scenario of selective processes due to the therapeutic and prophylactic use of antimicrobial agents, the processes of amplification of low level antimicrobial resistance (not usually detected or sought by common methods used for antibiotic resistance surveillance) might compromise the effectiveness of antibiotic chemotherapy.

Clinical outcome and risk factors related to extended-spectrum beta-lactamase-producing Klebsiella spp. infection among hospitalized patients

Over the past two decades, nosocomial infections caused by extended-spectrum beta-lactamase (ESBL)-producing Klebsiella spp. have become a major problem all around the world. This situation is of concern because there are limited antimicrobial options to treat patients infected with these pathogens, and also because this kind of resistance can spread to a wide variety of Gram-negative bacilli. Our objectives wereto evaluate among in-patients at a publicuniversity tertiary-care hospital with documented infection due to Klebsiella spp., which were the risk factors (cross-sectional analysis) and the clinical impact (prospective cohort) associated with an ESBL-producing strain. Study subjects were all patients admitted at the study hospital between April 2002 and October 2003, with a clinically and microbiologically confirmed infection caused by Klebsiella spp. at any body site, except infections restricted to the urinary tract. Of the 104 patients studied, 47 were infected with an ESBL-producing strain and 57 with a non-ESBL-producing strain. Independent risk factors associated with infection with an ESBL-producing strain were young age, exposure to mechanical ventilation, central venous catheter, use of any antimicrobial agent, and particularly use of a 4th generation cephalosporin or a quinolone. Length of stay was significant longer for patients infected with ESBL-producing strains than for those infected with non-ESBL-producing strains, although fatality rate was not significantly affected by ESBL-production in this cohort. In fact, mechanical ventilation and bacteremia were the only variables withindependent association withdeath detected in this investigation.

In vitro activity of fosfomycin against extended-spectrum-beta-lactamase-producing Escherichia coli and Klebsiella pneumoniae: comparison of susceptibility testing procedures

The agar dilution, broth microdilution, and disk diffusion methods were compared to determine the in vitro susceptibility of 428 extended-spectrum-beta-lactamase (ESBL)-producing Escherichia coli and Klebsiella pneumoniae to fosfomycin. Fosfomycin showed very high activity against all ESBL-producing strains. Excellent agreement between the three susceptibility methods was found for E. coli, whereas marked discrepancies were observed for K. pneumoniae.

blaCTX-M-2 and blaCTX-M-28 extended-spectrum β-lactamase genes and class 1 integrons in clinical isolates of Klebsiella pneumoniae from Brazil

Twenty-eight Klebsiella pneumoniae clinical isolates that exhibited an extended-spectrum cephalosporin-resistance profile from a city in the Northeast of Brazil were analysed by PCR and DNA sequencing in order to determine the occurrence of blaCTX-M genes and class 1 integrons. We determined the occurrence of the blaCTX-M-2 gene in six K. pneumoniae isolates and describe the first detection of the blaCTX-M-28 gene in South America. Seven isolates carried class 1 integrons. Partial sequencing analysis of the 5'-3'CS variable region in the class 1 integrons of three isolates revealed the presence of aadA1, blaOXA-2 and dfr22 gene cassettes.

Outbreak of carbapenem-resistant Klebsiella pneumoniae: two-year epidemiologic follow-up in a tertiary hospital

This study describes a carbapenem-resistant Klebsiella pneumoniae (CRKP) outbreak that occurred from October 2008-December 2010. Polymerase chain reaction assays were performed to detect the blaKPC gene and molecular typing was performed using pulsed-field gel electrophoresis (PFGE). There were 33 CRKP infections; PFGE revealed five genotypes: genotype A in five (15%), B in 18 (55%), C in eight (24%) and two unique profiles. Genotype B was disseminated in all hospital units and belonged to the same clone identified in 11 different hospitals in the state of São Paulo. Sixteen (48%) patients died. Seven isolates (21%) were resistant to polymyxin B and 45% were resistant to tigecycline and amikacin.

Whole genome sequence of Klebsiella pneumoniae U25, a hypermucoviscous, multidrug resistant, biofilm producing isolate from India

Klebsiella pneumoniae U25 is a multidrug resistant strain isolated from a tertiary care hospital in Chennai, India. Here, we report the complete annotated genome sequence of strain U25 obtained using PacBio RSII. This is the first report of the whole genome of K. pneumoniaespecies from Chennai. It consists of a single circular chromosome of size 5,491,870-bp and two plasmids of size 211,813 and 172,619-bp. The genes associated with multidrug resistance were identified. The chromosome of U25 was found to have eight antibiotic resistant genes [blaOXA-1,blaSHV-28, aac(6’)1b-cr,catB3, oqxAB, dfrA1]. The plasmid pMGRU25-001 was found to have only one resistant gene (catA1) while plasmid pMGRU25-002 had 20 resistant genes [strAB, aadA1,aac(6’)-Ib, aac(3)-IId,sul1,2, blaTEM-1A,1B,blaOXA-9, blaCTX-M-15,blaSHV-11, cmlA1, erm(B),mph(A)]. A mutation in the porin OmpK36 was identified which is likely to be associated with the intermediate resistance to carbapenems in the absence of carbapenemase genes. U25 is one of the few K. pneumoniaestrains to harbour clustered regularly interspaced short palindromic repeats (CRISPR) systems. Two CRISPR arrays corresponding to Cas3 family helicase were identified in the genome. When compared to K. pneumoniaeNTUHK2044, a transposase gene InsH of IS5-13 was found inserted.

Klebsiella pneumoniae strains producing extended-spectrum beta-lactamases in Spain: microbiological and clinical features.

Extended-spectrum β-lactamases (ESBL) of the CTX-M, SHV, and TEM families were recognized in 76 (67%), 31 (27%), and 6 (5%) isolates, respectively, among 162 ESBL-producing Klebsiella pneumoniae (ESBL-Kp) strains obtained in a multicenter study in Spain. Predisposing factors for ESBL-Kp acquisition included invasive procedures, mechanical ventilation, and previous antimicrobial use.

Vaccination of melanoma patients with Melan-A/Mart-1 peptide and Klebsiella outer membrane protein p40 as an adjuvant.

Toll-like receptor ligands are potentially useful adjuvants for the development of clinical T cell vaccination. Here we investigated the novel Toll-like receptor2 ligand P40, the outer membrane protein A derived from Klebsiella pneumoniae. Seventeen human leukocyte antigen-A*0201 positive stage III/IV melanoma patients were vaccinated with P40 and Melan-A/Mart-1 peptide subcutaneously in monthly intervals. Adverse reactions were mild-to-moderate. Fourteen patients received at least 8 vaccinations and were thus evaluable for clinical tumor and immune responses. Seven patients experienced progressive disease, whereas 2 patients had stable disease throughout the trial period, 1 of them with regression of multiple skin metastases. The remaining 5 patients had no measurable disease. Melan-A/Mart-1 specific CD8 T cells were analyzed ex vivo, with positive results in 6 of 14 evaluable patients. Increased percentages of T cells were found in three patients, memory/effector T cell differentiation in 4 patients, and a positive interferon-gamma Elispot assay in 1 patient. Antibody responses to P40 were observed in all patients. We conclude that vaccination with peptide and P40 was feasible and induced ex vivo detectable tumor antigen specific T cell responses in 6 of 14 patients with advanced melanoma.

New inhibitors of Helicobacter pylori urease holoenzyme selected from phage-displayed peptide libraries.

Urease is an important virulence factor for Helicobacter pylori and is critical for bacterial colonization of the human gastric mucosa. Specific inhibition of urease activity has been proposed as a possible strategy to fight this bacteria which infects billions of individual throughout the world and can lead to severe pathological conditions in a limited number of cases. We have selected peptides which specifically bind and inhibit H. pylori urease from libraries of random peptides displayed on filamentous phage in the context of pIII coat protein. Screening of a highly diverse 25-mer combinatorial library and two newly constructed random 6-mer peptide libraries on solid phase H. pylori urease holoenzyme allowed the identification of two peptides, 24-mer TFLPQPRCSALLRYLSEDGVIVPS and 6-mer YDFYWW that can bind and inhibit the activity of urease purified from H. pylori. These two peptides were chemically synthesized and their inhibition constants (Ki) were found to be 47 microM for the 24-mer and 30 microM for the 6-mer peptide. Both peptides specifically inhibited the activity of H. pylori urease but not that of Bacillus pasteurii.

Atividade da urease em latossolos sob influência da cobertura vegetal e da época de amostragem

O presente trabalho teve por objetivo avaliar o efeito do tipo de vegetação e da época de amostragem na atividade da urease em dois diferentes solos tropicais. O experimento foi instalado em Latossolo Vermelho Aluminoférrico típico e Latossolo Vermelho distrófico típico sob cinco diferentes culturas: pinus, eucalipto, citrus, soja e milho. As amostragens de solo foram efetuadas mensalmente, de abril de 1990 a março de 1991, determinando-se a atividade da urease, o N-total e o C-orgânico. A atividade da urease variou de acordo com a época de amostragem, apresentando valores mais elevados nos meses mais quentes e úmidos. A cobertura vegetal influenciou a conversão de N-uréia a N-NH4, observando-se maior atividade da urease nas amostras de solo sob pinus e eucalipto, embora, no início do ciclo das culturas da soja e do milho, a atividade da urease também tenha sido elevada.

Atividade de urease no solo com feijoeiro influenciada pela cobertura vegetal e sistemas de plantio

A urease é a enzima que catalisa a hidrólise da ureia em dióxido de carbono e amônia. A distribuição da urease e os fatores que a influenciam têm importância relevante em vista do uso da ureia na agricultura. Nesse sentido, este estudo teve como objetivo definir a época adequada de coleta de solo, após adubação nitrogenada no feijoeiro comum, para medir a atividade de urease e seu perfil, considerando os efeitos residuais de diferentes plantas de cobertura e de sistema de preparo do solo. O experimento foi instalado em um Latossolo Vermelho distrófico e o feijoeiro, cultivar BRS Valente, semeado em junho de 2005, em sucessão a quatro plantas de cobertura de solo: capim-mombaça, milho em consórcio com braquiária, sorgo granífero e estilosantes, e dois sistemas de cultivo, direto e convencional. O delineamento experimental foi o de blocos ao acaso em parcelas divididas. As amostras de solo foram coletadas aos 3, 6, 8, 10 e 12 dias após a aplicação da ureia em cobertura no feijoeiro. A maior atividade de urease no solo ocorreu quando o feijoeiro foi cultivado após capim-mombaça, independentemente do preparo do solo e da época de sua avaliação. A mobilização do solo em cultivo no sistema plantio direto determinou menor atividade de urease, independentemente das espécies de cobertura e das épocas de sua avaliação. O pico de atividade de urease ocorreu entre o sétimo e o oitavo dia após a aplicação de ureia, independentemente das espécies de cobertura e do preparo do solo sob cultivo irrigado do feijoeiro comum.

Volatilização de amônia do solo após a aplicação de ureia convencional ou com inibidor de urease

Volatilização de NH3 é a principal reação que diminui a eficiência de utilização pelas plantas do N proveniente da ureia, quando ela é aplicada sobre a superfície do solo. A fim de minimizar essa perda, produtos têm sido misturados à ureia para inibir temporariamente a ação da urease. Este trabalho objetivou avaliar alternativas de aplicação de um fertilizante com inibidor de urease, visando a diminuir a volatilização de NH3 relativamente à ureia convencional, em algumas condições ambientais e de solo. Foram desenvolvidos quatro experimentos, todos em condições de laboratório, em 2007 e 2008, em Cambissolo Húmico. Os tratamentos variaram em cada estudo e incluíram combinações de níveis de pH do solo (natural; 5,5; 6,3; e 6,8), umidade do solo (5, 10 ou 20 % de água) e temperaturas ambientais (18 e 35 ºC), além de estados físicos (sólido ou líquido) e de métodos de aplicação dos fertilizantes (na superfície ou incorporado ao solo). As unidades experimentais foram constituídas por bandejas plásticas (23 x 51 x 17 cm) com 12 kg de solo, numa espessura de 15 cm, sobre as quais foram instaladas câmaras coletoras de NH3. A amônia volatilizada foi determinada em várias épocas, nos primeiros 28 dias após a aplicação dos fertilizantes. O pico de volatilização diária de NH3 ocorreu sempre na primeira semana depois da adição dos fertilizantes ao solo, e aconteceu dois a três dias mais tarde para a ureia com inibidor de urease, em relação à ureia convencional. A volatilização de NH3 nem sempre foi maior para a ureia convencional em comparação ao fertilizante contendo inibidor de urease, tampouco para o estado líquido em relação ao granulado. A volatilização de NH3 aumentou com a elevação do pH, da temperatura e da dose aplicada de N e foi menor nos extremos de umidade (solo com 5 % ou com 20 % de água). Para os fertilizantes aplicados sobre a superfície do solo, a taxa máxima de perda diária foi correspondente a 14 kg ha-1 de N, e a perda total acumulada variou de 2 a 50 % do N aplicado, dependendo principalmente do estado físico em que o fertilizante foi aplicado, da umidade do solo e da temperatura ambiente. A incorporação dos fertilizantes amídicos ao solo foi a maneira mais eficaz de minimizar as perdas de N por volatilização.

Effects of nickel and nitrogen soil fertilization on lettuce growth and urease activity

Nickel is a micronutrient involved in nitrogen metabolism and a constituent of the urease molecule. Plant growth and urease activity were evaluated in lettuce (Lactuca sativa L.) grown in soil-filled pots in a 2 x 8 factorial design with two nitrogen (N) sources and eight Ni rates, with five replications. Nitrogen was applied at 200 mg dm-3 (half the dose incorporated into the soil at seedling transplanting and half top-dressed later) using the sources NH4NO3 (AN) and CO(NH2)2 (Ur). The Ni treatments (0, 2, 4, 8, 12, 16, 24 and 32 mg dm-3) were applied as NiCl2. The shoot dry-matter yield, leaf urease activity, Ni levels in the lettuce leaves and Ni levels extracted from soil with Mehlich-3 (M-3) and DTPA were determined. In the plants supplied with AN, the shoot dry-matter yield was higher than in those supplied with Ur. There was no difference in shoot dry matter in response to soil-applied Ni. The leaf urease activity increased with Ni application, regardless of the N source. The extractions with M-3 and DTPA were efficient to evaluate Ni availability for lettuce in the Red-Yellow Latosol.

Immunization of BALB/c mice with Helicobacter urease B induces a T helper 2 response absent in Helicobacter infection.

BACKGROUND & AIMS: Infection with Helicobacter induces a T helper type 1 response in mice and humans. Mice can be cured or protected from infection with Helicobacter by mucosal immunization with recombinant H. pylori urease B subunit (rUreB). This study characterizes the immune response of infected mice immunized with rUreB. METHODS: BALB/c mice were infected with H. felis. Two weeks later, they were orally immunized four times with rUreB and cholera toxin (CT) at weekly intervals. Controls were only infected or sham-immunized with CT. Animals were killed at various times after immunization. Splenic CD4(+) cells were obtained and cultured in vitro with rUreB to evaluate antigen-specific proliferation and induction of interferon gamma and interleukin 4 secretion. RESULTS: All rUreB-immunized mice (n = 8) were cured from infection 3 weeks after the fourth immunization. Immunization induced a proliferative response of splenic CD4(+) cells, a progressive decrease in interferon gamma secretion, and a concomitant increase in interleukin 4 secretion after each immunization. A simultaneous increase in rUreB specific serum immunoglobulin G1 levels was observed in infected/immunized mice. CONCLUSIONS: In BALB/c mice, therapeutic mucosal immunization with rUreB induces progressively a Th2 CD4(+) T cell response resulting in the elimination of the pathogen.