Evaluation of an adapted method of relative growth to determine the susceptibility of Enterobacterales to polymyxin B by MALDI-TOF MS.

Polymyxin B resistance is an emerging problem worldwide. The reference method to determine susceptibility to polymyxins is broth microdilution (BMD). As BMD is time consuming, it is necessary to develop new methodologies to provide faster evaluation of polymyxin susceptibility. This study aimed to evaluate polymyxin B susceptibility of Enterobacterales using an adapted methodology of relative growth (RG) by Matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS). A total of 60 isolates of Enterobacterales (22 resistant and 38 susceptible to polymyxin B by BMD) were evaluated. The adapted RG technique presented categorical agreement of 96.7% with only 2 major errors (3.3%) in comparison to BMD. Our findings demonstrate a high agreement between BMD and adapted RG, indicating that this methodology is promising for differentiating polymyxin B-susceptible isolates from polymyxin B-resistant isolates and could be implemented routinely in microbiology laboratories that already use the MALDI-TOF MS to identify bacteria.

Evaluation of filter paper as a means of transporting inactivated Gram-negative non-fermentative bacteria and Haemophilus spp. for identification using the MALDI-TOF MS system.

This study aimed to evaluate the filter paper as a means to transport inactivated Gram-negative non-fermentative (GNNF) bacteria and Haemophilus spp. for analysis using MALDI-TOF MS. A total of 133 isolates were evaluated and the analysis of each isolate was performed directly from original bacterial colony and in filter paper after the processing. To evaluate the agreement between the identification performed directly from the colony and after impregnation in filter paper, we assign the scores: >2·3 as excellent (E); 2·0 to 2·3 as very good (VG); 1·7-1·99 as good (G); <1·7 as unidentified (U). The divergences were classified as: Minor Divergence, Intermediate Divergence and Major Divergence. A total of 80 isolates transported in the filter paper disks presented full category concordance; 39 isolates presented Minor Divergence; 4 isolates present Intermediate Divergence; 4 isolates present Major Divergence and 6 isolates present better results after impregnation in filter paper. The proposed methodology of bacteria transportation presented a sensitivity of 96·9% and a specificity of 100%. The filter paper as a means to transport and storage of inactivated GNNF and Haemophilus spp. may be considered a potential tool for faster, more accurate, biosafe and less-expensive identification.

Blood metabolites and fecal starch as indicators of feed efficiency of beef cattle in the feedlot / [Metabolitos sanguíneos e amido fecal como indicadores da eficiência alimentar do gado de corte em confinamento]

The use of blood metabolites (BM), fecal starch (FS), and apparent digestion of starch, (ATTSD) as indicators of feed efficiency (FE) in beef cattle in the feedlot was studied. Fourteen bulls were used, originating in an industrial cross, without a defined racial group, with mean body weight of 284.86kg, individually fed, being evaluated in a 42-day confinement system. After the evaluation, the animals were divided into two groups according to the individual FE: high feed efficiency (HE) and low feed efficiency (LE). There was a difference between the groups in the variables FE, feed conversion (FC), final weight (FW), and daily weight gain (DWG). The FE had a positive correlation with DWG, FC, and FW. There was no difference between the groups for the variables BM, FS, and ATTSD, nor was there any correlation between these variables and FE. Considering the feed cost, the HE animals proved more profitable. BM, FS, and ATTSD did not statistically show potential to be used as indicators of FE, despite the evidence of numerical differences of these variables between the different groups, tendency of correlations with FE, and discriminating function with potential assertiveness.(AU)

Foi estudada a utilização dos metabólitos sanguíneos (BM), do amido fecal (FS) e da digestão aparente do amido (ATTSD) como indicadores de eficiência alimentar (FE) em bovinos de corte em confinamento. Utilizaram-se 14 touros, originários de cruzamento industrial, sem grupo racial definido, peso corporal médio de 284,86kg, alimentados individualmente, sendo avaliados em sistema de confinamento por 42 dias. Após a avaliação, dividiram-se os animais em dois grupos, de acordo com a FE individual: alta eficiência alimentar (HE) e baixa eficiência alimentar (LE). Houve diferença entre os grupos nas variáveis FE, conversão alimentar (FC), peso final (FW) e ganho de peso diário (DWG). A FE teve correlação positiva com DWG, FC e FW. Não houve diferença entre os grupos para as variáveis BM, FS e ATTSD, tampouco houve correlação entre essas variáveis e a FE. Considerando-se o custo alimentar, os animais HE mostraram-se mais lucrativos. BM, FS e ATTSD não mostraram, estatisticamente, potencial para serem utilizados como indicadores de FE, apesar da evidência de diferenças numéricas dessas variáveis entre os diferentes grupos, tendência de correlações com a FE e de função discriminante com potencial assertividade.(AU)

Evaluation of filter paper to transport inactivated bacteria to detect carbapenem resistance genes by multiplex real-time PCR using high-resolution melting.

Infections caused by resistant microorganisms are a complex global public health challenge, and the way to combat the increase of resistance is the development of more modern and faster techniques for resistance detection. This study aimed to evaluate the transport of inactivated bacteria impregnated in a filter paper disk to detect carbapenem resistance genes by multiplex real-time PCR (qPCR) using high-resolution melting (HRM). A total of 88 isolates of 10 different species of Enterobacterales harboring well-characterized carbapenem resistance genes were evaluated. A full 10-µL loop of fresh growth of bacteria were impregnated in a filter paper disk, which was left at room temperature for 2 days in order to simulate the time spent in transportation. Bacterial inactivation was performed with 70% ethanol at 15 min. Afterwards, the DNA was extracted from the paper disks for further analysis by qPCR HRM. The time of 15 min in 70% ethanol was enough to inactivate all the isolates tested. It was possible to correctly identify the presence of the carbapenem resistance gene by HRM qPCR in 87 isolates (98.87%) that were transported in the filter paper disks. Our results indicated that it is possible to use filter paper to transport inactivated bacteria and to identify carbapenem resistance genes by qPCR HRM. This alternative tends to facilitate the access to this technology by many laboratories which do not have the qPCR equipment.

Dissemination of bla OXA-370 gene among several Enterobacteriaceae species in Brazil.

OXA-370 is a recently described OXA-48 variant that has only been described in a few Enterobacter spp. and Klebsiella pneumoniae isolates. The purpose of this study is to assess the prevalence of OXA-370-producing isolates in carbapenem-nonsusceptible Enterobacteriaceae recovered from 28 hospitals from Brazil. Real-time PCR was used to determine the presence of bla NDM-1, bla KPC-2, bla VIM-type, bla GES-type, bla OXA-48-like, and bla IMP-type genes. A total of 4,451 Enterobacteriaceae were screened. The gene bla OXA-48-like was detected in 74 (2.5%) isolates, mostly of Enterobacter spp. (44.6% E. cloacae and 2.7% E. aerogenes) and Klebsiella spp. (31.1% K. pneumoniae and 6.7% K. oxytoca), followed by Escherichia coli, (6.7%), Morganella morganii, (2.7%), Citrobacter freundii (1.3%), Proteus mirabilis (1.3%), Providencia stuartii (1.3%), and Serratia spp. (1.3%). These isolates were from five hospitals, 67 (90.5%) from the hospital where the bla OXA-370 was first described. Sequencing of bla OXA-48-like was performed in 52 isolates, including E. cloacae, E. aerogenes, K. pneumoniae, K. oxytoca, E. coli, and C. freundii; all presenting 100% identity with bla OXA-370. PFGE revealed the presence of distinct clones among K. pneumoniae, E. cloacae, K. oxytoca, and E. coli. Susceptibility rates to meropenem, imipenem, and ertapenem among OXA-370-producing isolates were 92.3%, 78.8%, 7.7% respectively; the MIC50 /MIC90 were 0.38/2 mg/L and 1/3 mg/L for meropenem and imipenem respectively. Overall, antimicrobial susceptibility analysis suggests that OXA-370 lacks carbapenemase activity. Our study demonstrated that the bla OXA-370 gene is disseminated among several Enterobacteriaceae species and clones, indicating a high potential for dissemination.

Multi-state survey of healthcare-associated infections in acute care hospitals in Brazil.

BACKGROUND: Healthcare-associated infections (HCAIs) challenge public health in developing countries such as Brazil, which harbour social inequalities and variations in the complexity of healthcare and regional development. AIM: To describe the prevalence of HCAIs in hospitals in a sample of hospitals in Brazil. METHODS: A prevalence survey conducted in 2011-13 enrolled 152 hospitals from the five macro-regions in Brazil. Hospitals were classified as large (≥200 beds), medium (50-199 beds) or small sized (<50 beds). Settings were randomly selected from a governmental database, except for 11 reference university hospitals. All patients with >48 h of admission to the study hospitals at the time of the survey were included. Trained epidemiologist nurses visited each hospital and collected data on HCAIs, subjects' demographics, and invasive procedures. Univariate and multivariate techniques were used for data analysis. FINDINGS: The overall HCAI prevalence was 10.8%. Most frequent infection sites were pneumonia (3.6%) and bloodstream infections (2.8%). Surgical site infections were found in 1.5% of the whole sample, but in 9.8% of subjects who underwent surgical procedures. The overall prevalence was greater for reference (12.6%) and large hospitals (13.5%), whereas medium- and small-sized hospitals presented rates of 7.7% and 5.5%, respectively. Only minor differences were noticed among hospitals from different macro-regions. Patients in intensive care units, using invasive devices or at extremes of age were at greater risk for HCAIs. CONCLUSION: Prevalence rates were high in all geographic regions and hospital sizes. HCAIs must be a priority in the public health agenda of developing countries.

PCR to detect Mycobacterium tuberculosis in respiratory tract samples: evaluation of clinical data.

Tuberculosis (TB) remains as an important public health problem worldwide. Therefore, the rapid detection of M. tuberculosis is of primary importance to effectively reduce transmission in patients. The aims of this study were to evaluate two in-house molecular tests: nested PCR (nPCR) and real-time PCR (rtPCR) to detect M. tuberculosis complex directly from clinical samples. The results were compared to the culture results and to the culture results plus clinical data of patients. The rtPCR and nPCR presented high sensitivity (Se) and specificity (Sp) (rtPCR 97·6% and 91·5%, nPCR 85·7% and 92·7%, respectively) compared to culture. When the results of the molecular tests were compared to the culture plus clinical data the Se and Sp were 90·2% and 97·3% for rtPCR and 80·4% and 98·6% for the nPCR, respectively. The results demonstrated that molecular assays of M. tuberculosis can provide a sensitive and rapid diagnostic of TB, and when used in addition to the clinical data of TB patients will help to improve the Sp of the diagnosis of pulmonary TB.

Carbapenem-susceptible Acinetobacter baumannii carrying the ISAba1 upstream blaOXA-51-like gene in Porto Alegre, southern Brazil.

SUMMARY Over the last decade, Acinetobacter baumannii resistant to carbapenems has emerged in many medical centres and is commonly associated with high morbidity and mortality. We investigated potential mechanisms contributing to antimicrobial resistance of 58 clinical isolates of A. baumannii collected during a prolonged city-wide outbreak in five different hospitals in southern Brazil. The integrase gene was detected in 51 (87·9%) isolates of which 36 harboured class 2 integrons alone and 14 had both class 1 and 2 integrons; all carbapenem-resistant isolates displayed class 2 integrons. ISAba1 was found upstream of bla OXA-23-like only in isolates resistant to carbapenems; however, ISAba1 upstream of blaOXA-51-like was present in both susceptible and resistant isolates. This is the first report of a high prevalence of class 2 integrons in A. baumannii in southern Brazil. Moreover, our study suggests that ISAba1/blaOXA-51-like alone is insufficient to confer resistance to carbapenems.

A combined enrichment/polymerase chain reaction based method for the routine screening of Streptococcus agalactiae in pregnant women

Group B Streptococcus (GBS) is the most common cause of life-threatening infection in neonates. Guidelines from CDC recommend universal screening of pregnant women for rectovaginal GBS colonization. The objective of this study was to compare the performance of a combined enrichment/PCR based method targeting the atr gene in relation to culture using enrichment with selective broth medium (standard method) to identify the presence of GBS in pregnant women. Rectovaginal GBS samples from women at ¡Ý36 weeks of pregnancy were obtained with a swab and analyzed by the two methods. A total of 89 samples were evaluated. The prevalence of positive results for GBS detection was considerable higher when assessed by the combined enrichment/PCR method than with the standard method (35.9% versus 22.5%, respectively). The results demonstrated that the use of selective enrichment broth followed by PCR targeting the atr gene is a highly sensitive, specific and accurate test for GBS screening in pregnant women, allowing the detection of the bacteria even in lightly colonized patients. This PCR methodology may provide a useful diagnostic tool for GBS detection and contributes for a more accurate and effective intrapartum antibiotic and lower newborn mortality and morbidity.

A combined enrichment/polymerase chain reaction based method for the routine screening of Streptococcus agalactiae in pregnant women.

Group B Streptococcus (GBS) is the most common cause of life-threatening infection in neonates. Guidelines from CDC recommend universal screening of pregnant women for rectovaginal GBS colonization. The objective of this study was to compare the performance of a combined enrichment/PCR based method targeting the atr gene in relation to culture using enrichment with selective broth medium (standard method) to identify the presence of GBS in pregnant women. Rectovaginal GBS samples from women at ≥36 weeks of pregnancy were obtained with a swab and analyzed by the two methods. A total of 89 samples were evaluated. The prevalence of positive results for GBS detection was considerable higher when assessed by the combined enrichment/PCR method than with the standard method (35.9% versus 22.5%, respectively). The results demonstrated that the use of selective enrichment broth followed by PCR targeting the atr gene is a highly sensitive, specific and accurate test for GBS screening in pregnant women, allowing the detection of the bacteria even in lightly colonized patients. This PCR methodology may provide a useful diagnostic tool for GBS detection and contributes for a more accurate and effective intrapartum antibiotic and lower newborn mortality and morbidity.

When the resistance gets clingy: Pseudomonas aeruginosa harboring metallo-ß-lactamase gene shows high ability to produce biofilm.

The ability to produce biofilm and the presence of metallo-ß-lactamase (MBL) among Pseudomonas aeruginosa isolates were evaluated. A total of 91 isolates were recovered from sputa of patients with (CF, n = 44) and without (non-CF, n = 47) cystic fibrosis diagnosis. Seventy-nine (86.8%; 95% CI 78.3-92.3%) were biofilm producers. Interestingly, all isolates harboring MBL showed ability (most strong or moderate) to produce biofilm in vitro. We alert to an "overlapping of mechanisms" that together represent an even greater challenge for the treatment of pulmonary infections by P. aeruginosa.

A combined enrichment/polymerase chain reaction based method for the routine screening of Streptococcus agalactiae in pregnant women

Group B Streptococcus (GBS) is the most common cause of life-threatening infection in neonates. Guidelines from CDC recommend universal screening of pregnant women for rectovaginal GBS colonization. The objective of this study was to compare the performance of a combined enrichment/PCR based method targeting the atr gene in relation to culture using enrichment with selective broth medium (standard method) to identify the presence of GBS in pregnant women. Rectovaginal GBS samples from women at 36 weeks of pregnancy were obtained with a swab and analyzed by the two methods. A total of 89 samples were evaluated. The prevalence of positive results for GBS detection was considerable higher when assessed by the combined enrichment/PCR method than with the standard method (35.9% versus 22.5%, respectively). The results demonstrated that the use of selective enrichment broth followed by PCR targeting the atr gene is a highly sensitive, specific and accurate test for GBS screening in pregnant women, allowing the detection of the bacteria even in lightly colonized patients. This PCR methodology may provide a useful diagnostic tool for GBS detection and contributes for a more accurate and effective intrapartum antibiotic and lower newborn mortality and morbidity.

Evaluation of biofilm production by Pseudomonas Aeruginosa isolates recovered from cystic fibrosis and non-cystic fibrosis patients

Cystic fibrosis (CF) patients typically suffer of persistent and recurrent lung infections caused by Pseudomonas aeruginosa that many times possess ability for the biofilm production. Here, biofilm production among P. aeruginosa isolates recovered from sputum of CF and non-CF patients was evaluated. Most isolates were biofilm-producing independently of the patient's condition.

Evaluation of biofilm production by Pseudomonas Aeruginosa isolates recovered from cystic fibrosis and non-cystic fibrosis patients.

Cystic fibrosis (CF) patients typically suffer of persistent and recurrent lung infections caused by Pseudomonas aeruginosa that many times possess ability for the biofilm production. Here, biofilm production among P. aeruginosa isolates recovered from sputum of CF and non-CF patients was evaluated. Most isolates were biofilm-producing independently of the patient's condition.

Evaluation of biofilm production by Pseudomonas Aeruginosa isolates recovered from cystic fibrosis and non-cystic fibrosis patients

Cystic fibrosis (CF) patients typically suffer of persistent and recurrent lung infections caused by Pseudomonas aeruginosa that many times possess ability for the biofilm production. Here, biofilm production among P. aeruginosa isolates recovered from sputum of CF and non-CF patients was evaluated. Most isolates were biofilm-producing independently of the patient's condition.

Mycobacterium tuberculosis resistance in HIV-infected patients from a tertiary care teaching hospital in Porto Alegre, southern Brazil.

Drug-resistant Mycobacterium tuberculosis isolates are a major public health concern worldwide. There are few studies assessing tuberculosis (TB) resistance in Brazil. This study assessed the prevalence of resistance to the five first-line anti-TB drugs in TB isolates from HIV-infected patients in a tertiary care teaching hospital in southern Brazil. From September 1997 to July 2003, 398 TB complex isolates were included in the study. Resistance to one or more first-line anti-TB drugs was found in 71 (17.8%) of patients and was significantly more frequent among previously treated patients (12 [30.8%] of 39 patients) than new cases (59 [16.4%] of 359) (P=0.05). The highest resistance rates were found to isoniazid (9.9% overall; and 25.6% among previously treated patients). Multidrug-resistant TB was found in eight (2.0%) patients, with higher rates among previously treated patients than new cases: two (5.1%) patients vs. six (1.6%), respectively (P=0.18). Multidrug resistance and particularly isoniazid resistance rates among previously treated HIV patients are of great concern. Our findings indicate the need to reappraise regional TB treatment policies and support the recommendation for routine performance of in vitro TB susceptibility tests in all previously treated patients.

Dissemination of Pseudomonas aeruginosa producing SPM-1-like and IMP-1-like metallo-beta-lactamases in hospitals from southern Brazil.

BACKGROUND: Metallo-beta-lactamase (MBL) is an emerging resistance mechanism among Pseudomonas aeruginosa. The prevalence of this mechanism is particularly high in Latin America. We aimed to describe the prevalence and molecular characteristics of SPM-1-like, IMP-1-like and VIM type MBLs among ceftazidime and/or imipenem-resistant nosocomial P. aeruginosa isolates. METHODS: Pseudomonas aeruginosa isolates resistant to ceftazidime and/or imipenem recovered from hospitalized patients from two teaching hospitals from Porto Alegre, Brazil, were prospectively selected. Isolates were tested for MBL production using two phenotypic screening tests. Those isolates with positive results were further tested for the presence of MBL genes (SPM-1-like, IMP-1-like and VIM type) and submitted to molecular typing. RESULTS: A total of 92 isolates were analyzed and 33 (35.9%) were presumptively MBL producers by phenotypic tests. The SPM-1-like gene was found in 18 isolates and IMP-1-like in 5 isolates. In ten isolates the MBL type could not be identified. Three IMP-1-like isolates were susceptible to imipenem. SPM-1-like isolates comprised a single clone, and IMP-1-like isolates another single clone. CONCLUSION: The prevalence of MBL production among ceftazidime-resistant P. aeruginosa isolates is relatively high in both hospitals. Infection control measures have been challenged and further improvements in such measures are required to prevent dissemination of these isolates among hospitals. This is the first report of IMP-1-like MBLs in P. aeruginosa in southern Brazil.

Variations of agar screen tests for detection of methicillin resistance in staphylococci: focus on cefoxitin.

Members of the genus Staphylococcus are among the most important human pathogens, and strains demonstrating resistance to methicillin are an increasing problem worldwide, both within and outside of hospital environments. The objective of this study was to evaluate the use of variations of agar screening tests with cefoxitin and oxacillin to detect methicillin resistance in staphylococcal isolates. The agar screening test with cefoxitin (4 microg/ml) showed 99.4% accuracy for detecting both S. aureus and coagulase-negative staphylococci. The performance of the agar screening test with cefoxitin (4 microg/ml) either equaled or was superior to the other agar screening test variations evaluated and can be used to characterize the presence of the mecA gene among staphylococcal species.