Different virulence genetic context of multidrug-resistant CTX-M- and KPC-producing Klebsiella pneumoniae isolated from cerebrospinal fluid.

Information regarding resistance and virulence traits of meningitis-causing enterobacteria in hospital environment remains scarce. The aim of this study was to characterize virulence and acquired resistance genes of carbapenem-resistant and/or 3rd to 4th generation cephalosporin-resistant Klebsiella pneumoniae isolated from the cerebrospinal fluid of inpatients. Antimicrobial susceptibility testing was performed by disk diffusion. The string test was performed to identify hypermucoviscous phenotype. Galleria mellonella infection model was used to evaluate the virulence profile of the isolates. Screening for virulence determinants and acquired resistance genes were performed by PCR. The blaCTX-M and/or blaKPC and/or rmtG were detected in all the isolates. Genetic virulence determinants, including mrkD, entB, iroD, fecIRA, uge, wabG, fimH, ureA, ybtS, and clb were detected in the majority of multidrug-resistant K. pneumoniae isolates. One isolate presented hypermucoviscous phenotype, and several isolates showed enhanced virulence in G. mellonella infection model. The combination of the virulence genes found here seems to support not only the known virulence genetic context among nosocomial infections-causing K. pneumoniae but also the role that clb and ybtS may play in K. pneumoniae virulence.

Subinhibitory Concentrations of Biogenic Silver Nanoparticles Affect Motility and Biofilm Formation in Pseudomonas aeruginosa.

Biogenic silver nanoparticles (bio-AgNPs) are increasingly recognized as an antibiofilm and antivirulence strategy against P. aeruginosa, a bacterium that causes chronic infections in immunocompromised and cystic fibrosis patients. This study aimed to investigate the effects of subinhibitory concentrations of bio-AgNPs on motility and biofilm formation in P. aeruginosa. Bio-AgNPs were synthesized via reduction of ionic silver catalyzed by cell-free culture filtrate from Fusarium oxysporum. A total of 17 P. aeruginosa isolates and strains were evaluated for swarming, swimming, and twitching motility in the presence and absence (control) of bio-AgNPs, including 10 clinical isolates from patients with and without cystic fibrosis, 5 environmental isolates obtained from the public water supply system, and 2 reference strains (PAO1 and PA14). Isolates were identified by biochemical and molecular methods. Minimum inhibitory concentrations (MICs) were determined by the broth microdilution method. Swarming, swimming, and twitching motility assays were performed in Petri dishes. Biofilm formation capacity was assessed quantitatively by the crystal violet method. MIC values ranged from 15.62 to 62.50 µM. The results showed that subinhibitory concentrations of bio-AgNPs (½ MIC, 7.81-31.25 µM) significantly increased (p < 0.05) swarming, swimming, and twitching motility in 40.0, 40.0, and 46.7% of isolates, respectively. Subinhibitory bio-AgNP treatment enhanced (p < 0.05) biofilm formation capacity in PA14 and a cystic fibrosis isolate (P11). It is concluded that subinhibitory concentrations of bio-AgNPs increased biofilm formation and swarming, swimming, and twitching motility in PA14 and some P. aeruginosa isolates. These virulence factors are directly involved with quorum-sensing systems. Further research should investigate the effects of AgNPs on P. aeruginosa quorum sensing to help elucidate their mechanism of action at subinhibitory concentrations.

Draft genome sequence of an aminoglycoside-resistant RmtD2-producing Enterobacter cloacae subsp. cloacae ST395 in Brazil.

OBJECTIVES: Enterobacter cloacae has recently emerged as an important agent of nosocomial infections owing to the dissemination of extended-spectrum ß-lactamases and carbapenemases in this species. In this context, a rise in the therapeutic use of aminoglycosides was noticed, followed by the accelerated development of resistance mechanisms. In this study, we report the draft genome sequence of a multidrug-resistant E. cloacae subsp. cloacae strain (Ec2) isolated from an active surveillance culture of a patient with Chagas disease. METHODS: Whole-genome sequencing (WGS) was performed using a Nextera XT DNA library and NextSeq platform. RESULTS: WGS analysis revealed the presence of 5527 coding genes, 62 RNA-encoding genes and 275 pseudogenes. Strain Ec2 belongs to sequence type 395 (ST395) and carries 22 transferable antibiotic resistance genes, comprising eight antimicrobial classes, including the rmtD2 gene conferring high-level aminoglycoside resistance. CONCLUSIONS: This draft genome can be used in comparative genomic analyses with different E. cloacae strains. In addition, it could help at elucidating epidemiological aspects regarding the dissemination of clinically relevant resistance genes.

Evaluation of a new trilocus sequence-based multiplex-PCR to detect major Acinetobacter baumannii clonal complexes circulating in Brazil.

Dissemination of carbapenem-resistant Acinetobacter baumannii (CRAB) is mainly due to the spread of clonal lineages, particularly those included into the clonal complexes (CC) CC1, CC2, CC15, CC25, and CC79. We evaluated the usefulness of a recently modified PCR-based trilocus sequence-based typing (m3LST) in comparison with the standard multilocus sequence typing (MSLT) of 7 housekeeping genes as per the Institute Pasteur Scheme to assign the clonal complexes in CRAB. A collection of 78 CRAB isolated from 67 different Brazilian health institutions was submitted to both methodologies, and concordance rate was calculated. The collection studied included mainly isolates belonging to endemic Brazilian Clonal Complexes (CC1, CC15, CC25 and CC79, n=72, 92.3%) but also singletons sequence types (ST) with low prevalence in the country (ST107, ST113, ST188, ST317, ST584, ST733, n=6; 7.7%). The m3LST correctly assigned all the isolates into the main CC responsible for the CRAB dissemination in Brazil. All the singletons ST were not misidentified as prevalent lineages. The PCR-based m3LST is a powerful tool to investigate molecular epidemiology of A. baumannii representative of prevalent Brazilian clonal complexes 1, 15, 25 and 79.

Population Structure Analysis of Carbapenem-Resistant Acinetobacter baumannii Clinical Isolates from Brazil Reveals Predominance of Clonal Complexes 1, 15, and 79.

The population structure of 71 carbapenem-resistantAcinetobacter baumanniiclinical isolates from several hospitals in Brazil was investigated by ApaI pulsed-field gel electrophoresis,blaOXA-51-like subtyping, and multilocus sequence typing (Institute Pasteur scheme). In addition to the predominance of strains carryingblaOXA-23, we detected the presence ofblaOXA-72andblaOXA-231 We observed a predominance of clonal complex 1 (CC1), CC15, and CC79 and representative strains of the worldwide-disseminated international clone I.

Microbiological characterization of Delftia acidovorans clinical isolates from patients in an intensive care unit in Brazil.

Delftia acidovorans is an opportunistic agent in several types of infections, both in immunocompromised and immune-competent individuals; its resistance to aminoglycosides and polymyxin, choice drugs for empirical treatment of Gram-negative infections, is remarkable. We report the antimicrobial susceptibility and the genetic relatedness of 24 D. acidovorans strains recovered from tracheal aspirates of 21 adult inpatients hospitalized in an intensive care unit at a Brazilian hospital, from 2012 to 2013. All of the isolates were recovered as pure cultures and in counts above 1,000,000 CFU/mL. None of them were susceptible to polymyxin B, amikacin, gentamicin, or tobramycin; quinolones and trimethoprim-sulfamethoxazole presented varied activities against the isolates, while ß-lactam resistance was not detected. Four clusters were verified in pulsed-field gel electrophoresis analysis, and a major pulsotype comprised 10 strains. A possible, but undetermined common source, can be responsible for this strain dissemination, underscoring the need of reinforcing the adherence to disinfection and infection control standard techniques.

Effect that an educational program for cystic fibrosis patients and caregivers has on the contamination of home nebulizers.

OBJECTIVE: To describe the pathogens found in home nebulizers and in respiratory samples of cystic fibrosis (CF) patients, and to evaluate the effect that a standardized instruction regarding cleaning and disinfection of nebulizers has on the frequency of nebulizer contamination. METHODS: We included 40 CF patients (22 males), all of whom used the same model of nebulizer. The median patient age was 11.2 ± 3.74 years. We collected samples from the nebulizer mouthpiece and cup, using a sterile swab moistened with sterile saline. Respiratory samples were collected by asking patients to expectorate into a sterile container or with oropharyngeal swabs after cough stimulation. Cultures were performed on selective media, and bacteria were identified by classical biochemical tests. Patients received oral and written instructions regarding the cleaning and disinfection of nebulizers. All determinations were repeated an average of two months later. RESULTS: Contamination of the nebulizer (any part) was detected in 23 cases (57.5%). The nebulizer mouthpiece and cup were found to be contaminated in 16 (40.0%) and 19 (47.5%), respectively. After the standardized instruction had been given, there was a significant decrease in the proportion of contaminated nebulizers (43.5%). CONCLUSIONS: In our sample of CF patients, nebulizer contamination was common, indicating the need for improvement in patient practices regarding the cleaning and disinfection of their nebulizers. A one-time educational intervention could have a significant positive impact.

Effect that an educational program for cystic fibrosis patients and caregivers has on the contamination of home nebulizers / Efeito de um programa de educação para cuidadores e pacientes com fibrose cística na contaminação de nebulizadores de uso domiciliar

OBJECTIVE: To describe the pathogens found in home nebulizers and in respiratory samples of cystic fibrosis (CF) patients, and to evaluate the effect that a standardized instruction regarding cleaning and disinfection of nebulizers has on the frequency of nebulizer contamination. METHODS: We included 40 CF patients (22 males), all of whom used the same model of nebulizer. The median patient age was 11.2 ± 3.74 years. We collected samples from the nebulizer mouthpiece and cup, using a sterile swab moistened with sterile saline. Respiratory samples were collected by asking patients to expectorate into a sterile container or with oropharyngeal swabs after cough stimulation. Cultures were performed on selective media, and bacteria were identified by classical biochemical tests. Patients received oral and written instructions regarding the cleaning and disinfection of nebulizers. All determinations were repeated an average of two months later. RESULTS: Contamination of the nebulizer (any part) was detected in 23 cases (57.5%). The nebulizer mouthpiece and cup were found to be contaminated in 16 (40.0%) and 19 (47.5%), respectively. After the standardized instruction had been given, there was a significant decrease in the proportion of contaminated nebulizers (43.5%). CONCLUSIONS: In our sample of CF patients, nebulizer contamination was common, indicating the need for improvement in patient practices regarding the cleaning and disinfection of their nebulizers. A one-time educational intervention could have a significant positive impact. .

OBJETIVO: Descrever os patógenos encontrados nos nebulizadores de uso domiciliar e nas amostras de trato respiratório de pacientes com fibrose cística (FC) e verificar o efeito de uma instrução padronizada de higiene e desinfecção de nebulizadores na contaminação dos mesmos. MÉTODOS: Foram incluídos no estudo 40 pacientes com FC (22 do sexo masculino) que utilizavam um mesmo modelo de nebulizador. A mediana de idade foi de 11,2 ± 3,74 anos. Amostras dos nebulizadores foram coletadas do bocal e do copo reservatório utilizando-se um swab estéril umedecido em solução salina estéril. As amostras de trato respiratório dos pacientes foram colhidas por expectoração em coletor estéril ou com swab de orofaringe após estímulo de tosse. As culturas foram realizadas em meios seletivos, e a identificação bacteriana foi feita através de provas bioquímicas clássicas. Instruções verbais e escritas sobre higiene e desinfecção dos nebulizadores foram ministradas. Todas as determinações foram repetidas dois meses após, em média. RESULTADOS: A contaminação de alguma parte dos nebulizadores foi observada em 23 casos (57,5%). A contaminação do bocal e do copo foi similar, em 16 (40.0%) e 19 casos (47.5%), respectivamente. Houve uma redução significativa da proporção de nebulizadores contaminados (43,5%) após a instrução padronizada. CONCLUSÕES: Nesta amostra de pacientes com FC, a contaminação dos nebulizadores foi alta, o que indica a necessidade de melhoria nas práticas de higiene e desinfecção dos nebulizadores de pacientes com FC. Uma única intervenção educacional pode ter um impacto positivo significativo. .

Nosocomial infections caused by Elizabethkingia meningoseptica: an emergent pathogen.

We hereby describe the clinical and epidemiological features and, outcomes of nine patients with Elizabethkingia meningoseptica infections in two hospitals over a 2-year period. All infections caused by this pathogen were nosocomial, or healthcare associated infections, in hemodialysis settings whereas none was correlated with hospital outbreaks.

The combination of PCR and serology increases the diagnosis of Pseudomonas aeruginosa colonization/infection in cystic fibrosis.

BACKGROUND: Early diagnosis of Pseudomonas aeruginosa colonization/infection in patients with cystic fibrosis (CF) using microbiological culturing methods may be difficult. Serology and polymerase chain reaction (PCR) may be useful techniques for early detection of P. aeruginosa in children with CF. METHODS: A cross-sectional analysis comparing results obtained by three different methods for P. aeruginosa identification was performed in 87 CF patients with a mean age of 9.7 years. Microbiological culturing and PCR targeting the algD GDP mannose dehydrogenase gene of P. aeruginosa were performed in sputum or oropharyngeal swabs samples, and serum antibodies against three P. aeruginosa antigens (elastase, alkaline protease, and exotoxin A) were assessed once. RESULTS: It was possible to isolate P. aeruginosa by culture in samples from 42 patients (48.2%), while PCR was positive in 53 (60.9%) patients. Serology was positive in 38 patients (43.6%), with a higher positivity for elastase (37.9%), followed by alkaline protease (29.9%) and exotoxin A (19.5%). The difference among the three isolated methods was not statistically significant. The combination of PCR + serology was significantly superior to single methods, to PCR + culture and also to culture + serology. CONCLUSIONS: PCR identified a higher number of patients with P. aeruginosa than serology and conventional culture, but the difference did not reach statistical significance. Any of the combination methods that included PCR resulted in significantly statistical differences in relation to isolated microbiological or serology methods, but not to the PCR method alone, suggesting that PCR may be the main additive method for P. aeruginosa identification.

Proteases (caseinase and elastase), hemolysins, adhesion and susceptibility to antimicrobials of Stenotrophomonas maltophilia isolates obtained from clinical specimens

Forty-six S. maltophilia isolates obtained from hospital clinical specimens were studied for protease (caseinase and elastase) production, hemolytic activity, adhesion to HEp-2 cells, plastic and glass. Susceptibility to antimicrobial agents was also evaluated. The majority of isolates were obtained from respiratory tract secretions of patients using medical devices. All the isolates grown overnight were able to hydrolyze casein at 30ºC and 37ºC. After 72h, all the isolates hydrolyzed elastase at 30ºC and 40 isolates (87 per cent) at 37ºC. Most of the isolates presented hemolytic activity after 96h of incubation at both temperatures. Rabbit blood showed the hightest hemolytic activity, after 96h 61(per cent) and 98(per cent) of tested isolates presented b-hemolysis at 30ºC and 37ºC, respectively. All isolates were susceptible to trimethoprim-sulfametoxazole and were resistant to most b-lactams tested. By the dilution method, S. maltophilia showed a high susceptibility to ticarcillin-clavulanate and a lower susceptibility to ciprofloxacin than the agar diffusion. The isolates showed adhesion to HEp-2 cells, plastic and glass. The proteolytic activities and adhesion to inanimate surfaces detected in S. maltophilia can be related to the pathogenesis of this bacterium and/or medical device colonization which favors the development of nosocomial infections.