Antiseptic management of critical wounds: differential bacterial response upon exposure to antiseptics and first insights into antiseptic/phage interactions - experimental studies.

BACKGROUND: With the antibiotic crisis, the topical antibacterial control including chronic wounds gains increasing importance. However, little is known regarding tolerance development when bacteria face repetitive exposure to the identical antiseptics as commonly found in clinical practice. MATERIALS AND METHODS: We exposed clinical isolates foremost of chronic wounds in vitro to dilutions of two antisepctics used for wound therapy: polyhexanide or octenidine. Adaptive response was determined by growth/kill curves, minimal inhibitory concentration (MIC), and whole genome sequencing. Antiseptic/bacteriophage combinations were studied by liquid-infection assays and bacterial plating. RESULTS: Polyhexanide acted stronger against Escherichia coli and Proteus mirabilis while octenidine was more potent against Staphylococcus aureus. Otherwise, the antiseptic efficacy varied across isolates of Klebsiella pneumoniae, Pseudomonas aeruginosa, and Acinetobacter baumannii. Upon repetitive exposure with constant antiseptic concentrations P. aeruginosa and P. mirabilis adaptation was evident by a reduced lag-phase and a two-fold increased MIC. Under increasing octenidine concentrations, P. aeruginosa adapted to an eightfold higher dosage with mutations in smvA, opgH and kinB affecting an efflux pump, alginate and biofilm formation, respectively. S. aureus adapted to a fourfold increase of polyhexanide with a mutation in the multiple peptide resistance factor MprF, also conferring cross-resistance to daptomycin. Antiseptic/bacteriophage combinations enhanced bacterial inhibition and delayed adaptation. CONCLUSION: Different bacterial species/strains respond unequally to low-level antiseptic concentrations. Bacterial adaptation potential at phenotypic and genotypic levels may indicate the necessity for a more nuanced selection of antiseptics. Bacteriophages represent a promising yet underexplored strategy for supporting antiseptic treatment which may be particularly beneficial for the management of critical wounds.

Enlarging the Toolbox Against Antimicrobial Resistance: Aptamers and CRISPR-Cas.

In the post-genomic era, molecular treatments and diagnostics have been envisioned as powerful techniques to tackle the antimicrobial resistance (AMR) crisis. Among the molecular approaches, aptamers and CRISPR-Cas have gained support due to their practicality, sensibility, and flexibility to interact with a variety of extra- and intracellular targets. Those characteristics enabled the development of quick and onsite diagnostic tools as well as alternative treatments for pan-resistant bacterial infections. Even with such potential, more studies are necessary to pave the way for their successful use against AMR. In this review, we highlight those two robust techniques and encourage researchers to refine them toward AMR. Also, we describe how aptamers and CRISPR-Cas can work together with the current diagnostic and treatment toolbox.

Exploring the Potential of CRISPR-Cas9 Under Challenging Conditions: Facing High-Copy Plasmids and Counteracting Beta-Lactam Resistance in Clinical Strains of Enterobacteriaceae.

The antimicrobial resistance (AMR) crisis urgently requires countermeasures for reducing the dissemination of plasmid-borne resistance genes. Of particular concern are opportunistic pathogens of Enterobacteriaceae. One innovative approach is the CRISPR-Cas9 system which has recently been used for plasmid curing in defined strains of Escherichia coli. Here we exploited this system further under challenging conditions: by targeting the bla TEM- 1 AMR gene located on a high-copy plasmid (i.e., 100-300 copies/cell) and by directly tackling bla TEM- 1-positive clinical isolates. Upon CRISPR-Cas9 insertion into a model strain of E. coli harboring bla TEM- 1 on the plasmid pSB1A2, the plasmid number and, accordingly, the bla TEM- 1 gene expression decreased but did not become extinct in a subpopulation of CRISPR-Cas9 treated bacteria. Sequence alterations in bla TEM- 1 were observed, likely resulting in a dysfunction of the gene product. As a consequence, a full reversal to an antibiotic sensitive phenotype was achieved, despite plasmid maintenance. In a clinical isolate of E. coli, plasmid clearance and simultaneous re-sensitization to five beta-lactams was possible. Reusability of antibiotics could be confirmed by rescuing larvae of Galleria mellonella infected with CRISPR-Cas9-treated E. coli, as opposed to infection with the unmodified clinical isolate. The drug sensitivity levels could also be increased in a clinical isolate of Enterobacter hormaechei and to a lesser extent in Klebsiella variicola, both of which harbored additional resistance genes affecting beta-lactams. The data show that targeting drug resistance genes is encouraging even when facing high-copy plasmids. In clinical isolates, the simultaneous interference with multiple genes mediating overlapping drug resistance might be the clue for successful phenotype reversal.

Fighting Pathogenic Bacteria on Two Fronts: Phages and Antibiotics as Combined Strategy.

With the emerging threat of infections caused by multidrug resistant bacteria, phages have been reconsidered as an alternative for treating infections caused by tenacious pathogens. However, instead of replacing antibiotics, the combination of both types of antimicrobials can be superior over the use of single agents. Enhanced bacterial suppression, more efficient penetration into biofilms, and lowered chances for the emergence of phage resistance are the likely advantages of the combined strategy. While a number of studies have provided experimental evidence in support of this concept, negative interference between phages and antibiotics have been reported as well. Neutral effects have also been observed, but in those cases, combined approaches may still be important for at least hampering the development of resistance. In any case, the choice of phage type and antibiotic as well as their mixing ratios must be given careful consideration when deciding for a dual antibacterial approach. The most frequently tested bacterium for a combined antibacterial treatment has been Pseudomonas aeruginosa, but encouraging results have also been reported for Escherichia coli, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Enterococcus faecalis, and Burkholderia cepacia. Given the immense play area of conceivable phage-antibiotic combinations and their potential excess value, it is time to recapitulate of what has been achieved so far. This review therefore gathers and compares the results from most relevant studies in order to help researchers and clinicians in their strategies to combat multidrug resistant bacteria. Special attention is given to the selected bacterial model organisms, the phage families and genera employed, and the experimental design and evaluation (e.g., in vitro vs. in vivo models, biofilm vs. planktonic culture experiments, order and frequency of administration etc.). The presented data may serve as a framework for directed further experimental approaches to ultimately achieve a resolute challenge of multidrug resistant bacteria based on traditional antibiotics and phages.

Investigating cross-contamination by yeast strains from dental solid waste to waste-handling workers by DNA sequencing.

Trying to widen the discussion on the risks associated with dental waste, this study proposed to investigate and genetically compare yeast isolates recovered from dental solid waste and waste workers. Three samples were collected from workers' hands, nasal mucosa, and professional clothing (days 0, 30, and 180), and two from dental waste (days 0 and 180). Slide culture, microscopy, antifungal drug susceptibility, intersimple sequence repeat analysis, and amplification and sequencing of internal transcribed spacer regions were performed. Yeast strains were recovered from all waste workers' sites, including professional clothes, and from waste. Antifungal susceptibility testing demonstrated that some yeast recovered from employees and waste exhibited nonsusceptible profiles. The dendrogram demonstrated the presence of three major clusters based on similarity matrix and UPGMA grouping method. Two branches displayed 100% similarity: three strains of Candida guilliermondii isolated from different employees, working in opposite work shifts, and from diverse sites grouped in one part of branch 1 and cluster 3 that included two samples of Candida albicans recovered from waste and the hand of one waste worker. The results suggested the possibility of cross-contamination from dental waste to waste workers and reinforce the need of training programs focused on better waste management routines.

Knowledge, behaviour and microbial load of workers handling dental solid waste in a public health service in Brazil.

Healthcare wastes are those generated inside healthcare services, including dental clinics. Workers coming into close proximity to hazardous healthcare waste are potentially at risk. In an attempt to assess the knowledge and attitudes of workers dealing with infectious waste, a questionnaire was administered. The biological risk was investigated by evaluating the microbial load and screening some clinically relevant micro-organisms in the nasal mucosa, hands and coats of these workers. The results showed that 66.6% of the study population had incomplete primary education. Only two workers have had their blood tested to confirm anti-HBs levels. Microbial load evaluation on hand surfaces of morning workers showed statistically significant lower microbial loads after the workday when compared with the beginning of the work period. It is important to highlight that some clinically relevant bacteria, such as Pseudomonas aeruginosa and Klebsiella pneumoniae were isolated from worker's hands. This study revealed the need for more training programmes regarding awareness of safe waste disposal protocols and also the necessity of discussing vaccination and its implications. Data regarding microbial loads of the worker's hands, mostly at the beginning of the workday when handwashing is recommended worldwide, emphasise that hygiene measures should receive more attention during training exercises.

Test-retest reliability and concurrent validity of a digital manovacuometer / La reproducibilidad de test-retest y la validez concurrente del manovacuometro digital / Reprodutibilidade teste-reteste e validade concorrente de manovacuômetro digital

The manovacuometer is a simple, quick and non-invasive test which measures the maximal respiratory pressures (MRS). Guidelines recommend the use of a digital manovacuometer due to its high accuracy. The purpose of this study was to assess the test-retest reliability and concurrent validity of a digital manovacuometer in measuring the maximal inspiratory and expiratory pressures (MIP/MEP) and nasal inspiratory pressure while sniffing (SNIP). A total of 30 healthy subjects were assessed (20-30 years old) using the UFMG and MicroRPM(r) (Micro Medical, UK) digital manovacuometers. To assess reliability, Intraclass Correlation Coefficient (ICC) and Student's t test it was used for dependent samples. For the validity assessment, the following were used: Pearson correlation, Student's t test for dependent samples, linear regression and the Bland-Altman method. The level of significance was set at 5% (p<0.05). The ICC values were significant and showed a good magnitude (0.76 to 0.89) and no significant differences were found between the means of the variables of the UFMG digital manovacuometer analyzed within two days (p>0.05); the correlation between observed values from the two instruments was of high magnitude for all variables (0.82 to 0.85); no significant difference was found between the values obtained for both instruments (p>0.05); a strong association was observed between measures of MIP and MEP obtained by the two methods and Bland-Altman analysis showed no systematic overestimation or underestimation of maximal respiratory pressures and SNIP. In conclusion, the results suggest that the UFMG manovacuometer is a reliable and valid instrument for assessing MIP, MEP and SNIP in healthy subjects.

La manovacuometría es una prueba sencilla, rápida y no invasiva que mide las presiones respiratorias máximas (PRM). Directrices recomiendan el uso del manuvacuómetro digital debido a su alta precisión. El objetivo de este estudio fue evaluar la reproducibilidad test-retest y la validez concurrente de un manuvacuómetro digital para medir las presiones inspiratoria y espiratoria máximas (PImáx y PEmáx) y de la presión inspiratoria nasal durante la aspiración (SNIP). Se evaluaron 30 sujetos sanos (20-30 años) por medio de los manovacuómetros digitales UFMG y MicroRPM(r) (Micro Medical, UK). Para evaluar la reproducibilidad, se utilizó el coeficiente de correlación intraclase (CCI) y el test t de student para muestras dependientes. Para el análisis de la validez se utilizaron: la correlación de Pearson, el test t de student para muestras dependientes, el análisis de regresión lineal y el método Bland-Altman. El nivel de significación considerado fue del 5% (p<0,05). Los valores de CCI fueron significativos y de buena magnitud (0,76 a 0,89) y no se encontraron diferencias significativas entre las medias de las variables del manovacuómetro UFMG analizadas en los dos días (p>0,05). La correlación entre los valores observados en los dos instrumentos fue de alta magnitud para todas las variables (0,82 a 0,85); no hubo diferencia significativa entre los valores medios obtenidos en los dos instrumentos (p>0,05); Se observó una fuerte asociación entre las medidas de las PRM obtenidas por los dos métodos y el análisis de Bland-Altman no demostró sobreestimación o subestimación sistemática de las PRM y del SNIP. En conclusión, los resultados sugieren que el manovacuómetro UFMG es fiable y válido para la evaluación de las PRM y SNIP en sujetos sanos.

A manovacuometria é um teste simples, rápido e não invasivo que mensura as pressões respiratórias máximas (PRM). Diretrizes recomendam o uso do manovacuômetro digital devido à sua alta precisão. O objetivo deste estudo foi avaliar a reprodutibilidade teste-reteste e a validade concorrente de um manovacuômetro digital na mensuração das pressões inspiratórias e expiratórias máximas (PImáx e PEmáx) e da pressão inspiratória nasal durante o fungar (SNIP). Foram avaliados 30 indivíduos saudáveis (20-30 anos) utilizando os manovacuômetros digitais UFMG e MicroRPM(r) (Micro Medical, UK). Para avaliar a reprodutibilidade, foi utilizado o Coeficiente de Correlação Intraclasse (CCI) e teste t de student para amostras dependentes. Para análise da validade foram utilizados: a correlação de Pearson, o teste t de student para amostras dependentes, a análise de regressão linear e o método Bland-Altman. O nível de significância considerado foi de 5% (p<0,05). Os valores de CCI foram significativos e de boa magnitude (0,76 a 0,89) e não foram encontradas diferenças significativas entre as médias das variáveis do manovacuômetro UFMG analisadas nos dois dias (p>0,05). A correlação entre os valores observados nos dois instrumentos foi de alta magnitude para todas as variáveis (0,82 a 0,85); não houve diferença significativa entre os valores médios obtidos nos dois instrumentos (p>0,05); foi observada forte associação entre as medidas das PRM obtidas pelos dois métodos e a análise de Bland-Altman não demonstrou superestimação ou subestimação sistemática das PRM e do SNIP. Em conclusão, os resultados sugerem que o manovacuômetro UFMG é confiável e válido para avaliação das PRM e SNIP em indivíduos saudáveis.

A avaliação do desenvolvimento infantil: um desafio interdisciplinar / Evaluation of childhood development: an interdisciplinary challenge

OBJETIVO: Avaliar o desenvolvimento de crianças de 2 meses a 2 anos de idade por meio da Atenção Integrada às Doenças Prevalentes na Infância (AIDPI), no contexto do Programa de Educação pelo Trabalho em Saúde (PET-Saúde). MÉTODO: Estudo transversal realizado com 122 crianças, com idades entre 2 meses e 2 anos, da área de abrangência do Centro de Saúde São Bernardo (CSSB) - Belo Horizonte (MG), em 2009. Os dados relativos ao desenvolvimento foram obtidos através da aplicação de dois questionários: AIDPI e Caderneta de Saúde da Criança (CSC). Foram comparadas as classificações do desenvolvimento pela AIDPI e pela CSC, a associação entre atraso do desenvolvimento e as variáveis estudadas. RESULTADOS: As características com maior frequência na população estudada foram a baixa escolaridade das mães (62,1%), seguida de parentes com deficiência mental (71,3%) e problemas na gestação (71,3%). A AIDPI evidenciou que 61,5% da população estudada encontra-se normal com fator de risco, 16,4% normal sem fator de risco, 11,5% com possível atraso e 10,7% com provável atraso do desenvolvimento infantil. A concordância observada entre a classificação da AIDPI e da CSC foi de 0,34, coeficiente Kappa igual a - 0,12 (p = 0,98). Não houve associação estatisticamente significativa entre as variáveis analisadas (frequenta creches; convívio com problemas emocionais; escolaridade da mãe; idade gestacional; e peso ao nascer) e atraso possível/provável do desenvolvimento identificado pela AIDPI. CONCLUSÃO: O PET-Saúde, como proposta de integração da educação pelo trabalho, permitiu uma oportunidade de convivência e troca de experiências entre alunos e profissionais de diferentes áreas de atuação, trabalhando em um projeto comum.

OBJECTIVE: The aim of this study was to evaluate early childhood development, from two months to two years of age, based on Integrated Management of Childhood Illness, IMCI (or AIDPI in Portuguese) in the context of the Educational Program for Health Work (PET-Saúde). METHODS: A cross-sectional study was conducted with 122 children two months to two years of age from the coverage area of the São Bernardo Health Center in Belo Horizonte, Minas Gerais State, in 2009. Data on the children´s development were obtained using two questionnaires: IMCI and the Children's Health Booklet (CSC in Portuguese). Early childhood development according to the IMCI classification was compared to the results with CSC. The authors also investigated potential associations between independent variables and developmental delay. RESULTS: The most frequent characteristics in the study population were low maternal schooling (62.1%), followed by relatives with mental disability (71.3%), and problems during the pregnancy (71.3%). Based on the IMCI classification, 61.5% of the study population was normal with some risk factor, 16.4% normal without any risk factor, 11.5% with possible delay, and 10.7% with probable childhood developmental delay. Agreement between the IMCI and CSC classifications was 0.34, kappa index - 0.12 (p = 0.98). There was no statistically significant association between the variables (daycare attendance; contact with emotional problems; maternal schooling; gestational age; and birth weight) and possible or probable early childhood developmental delay according to the IMCI classification. CONCLUSION: PET-Saúde, as a proposal for integration between education and work, provided an opportunity for contact and exchange of experiences between students and health professionals from various fields, working in a common project.