Report and Comparative Genomics of an NDM-5-Producing Escherichia coli in a Portuguese Hospital: Complex Class 1 Integrons as Important Players in blaNDM Spread.

BACKGROUND: New Delhi metallo-beta-lactamase (NDM) has been spreading across the globe, but the causes of its success are poorly understood. We characterized a blaNDM-5-positive Escherichia coli strain from a Portuguese hospital and conducted comparative genomic analyses to understand the role of clonal background and horizontal gene transfer in blaNDM-5 dissemination. METHODS: After blaNDM PCR screening and genome sequencing, Ec355340 was subjected to mating, transformation, and plasmid curing assays and MICs determination for several antibiotics. Comparison with data compiled from public databases was performed. RESULTS: blaNDM-5 was in a complex integron co-located in a FIB-FII plasmid (pEc355340_NDM-5). The mating assays were unsuccessful, but plasmid transformation into a susceptible host led to resistance to all beta-lactams and to sulfamethoxazole-trimethoprim. The profile of virulence genes (n = 73) was compatible with extraintestinal pathogenesis. An analysis of genomes from public databases suggested that blaNDM-5 has rarely been associated with ST156 strains (such as Ec355340), while is has frequently been found on strains of the ST10 clonal complex. However, ST156 may play a role in the co-spreading of blaNDM and mcr genes. Regardless, comparative genomics confirmed the presence of blaNDM in similar complex integrons in plasmids (48/100 plasmids most similar to pEc355340_NDM-5) and ST156 genomes (20/41 blaNDM-positive genomes). CONCLUSIONS: blaNDM-5 and other blaNDM variants were more frequently associated to complex integrons than previously reported and, therefore, these platforms may be important drivers in their dissemination. The identification of blaNDM-5 for the first time in Portugal could be a game-changer in the current Portuguese antibiotic resistance scenario, as this gene encodes a higher-level resistance phenotype, and its spread may be facilitated due to the association with complex integrons.

In vitro activity of imipenem/relebactam against Pseudomonas aeruginosa isolates recovered from ICU patients in Spain and Portugal (SUPERIOR and STEP studies).

OBJECTIVES: To study the in vitro activity of imipenem/relebactam and comparators and the imipenem/relebactam resistance mechanisms in a Pseudomonas aeruginosa collection from Portugal (STEP, 2017-18) and Spain (SUPERIOR, 2016-17) surveillance studies. METHODS: P. aeruginosa isolates (n = 474) were prospectively recovered from complicated urinary tract (cUTI), complicated intra-abdominal (cIAI) and lower respiratory tract (LRTI) infections in 11 Portuguese and 8 Spanish ICUs. MICs were determined (ISO broth microdilution). All imipenem/relebactam-resistant P. aeruginosa isolates (n = 30) and a subset of imipenem/relebactam-susceptible strains (n = 32) were characterized by WGS. RESULTS: Imipenem/relebactam (93.7% susceptible), ceftazidime/avibactam (93.5% susceptible) and ceftolozane/tazobactam (93.2% susceptible) displayed comparable activity. The imipenem/relebactam resistance rate was 6.3% (Portugal 5.8%; Spain 8.9%). Relebactam restored imipenem susceptibility to 76.9% (103/134) of imipenem-resistant isolates, including MDR (82.1%; 32/39), XDR (68.8%; 53/77) and difficult-to-treat (DTR) isolates (67.2%; 45/67). Among sequenced strains, differences in population structure were detected depending on the country: clonal complex (CC)175 and CC309 in Spain and CC235, CC244, CC348 and CC253 in Portugal. Different carbapenemase gene distributions were also found: VIM-20 (n = 3), VIM-1 (n = 2), VIM-2 (n = 1) and VIM-36 (n = 1) in Spain and GES-13 (n = 13), VIM-2 (n = 3) and KPC-3 (n = 2) in Portugal. GES-13-CC235 (n = 13) and VIM type-CC175 (n = 5) associations were predominant in Portugal and Spain, respectively. Imipenem/relebactam showed activity against KPC-3 strains (2/2), but was inactive against all GES-13 producers and most of the VIM producers (8/10). Mutations in genes affecting porin inactivation, efflux pump overexpression and LPS modification might also be involved in imipenem/relebactam resistance. CONCLUSIONS: Microbiological results reinforce imipenem/relebactam as a potential option to treat cUTI, cIAI and LRTI caused by MDR/XDR P. aeruginosa isolates, except for GES-13 and VIM producers.

Imipenem-Relebactam Susceptibility in Enterobacterales Isolates Recovered from ICU Patients from Spain and Portugal (SUPERIOR and STEP Studies).

Imipenem-relebactam is a novel ß-lactam-ß-lactamase inhibitor combination. We evaluated the in vitro activity of imipenem-relebactam and comparators against Enterobacterales clinical isolates recovered in 8 Spanish and 11 Portuguese intensive care units (ICUs) (SUPERIOR, 2016-2017; STEP, 2017-2018). Overall, 747 Enterobacterales isolates (378 Escherichia coli, 252 Klebsiella spp., 64 Enterobacter spp., and 53 other species) were prospectively collected from ICU patients with complicated intraabdominal (cIAI), complicated urinary tract (cUTI), and lower respiratory tract (LRTI) infections. MICs were determined (ISO-broth microdilution), and whole-genome sequencing (WGS) was performed in a subset of isolates displaying susceptible and resistant imipenem-relebactam MICs. Imipenem-relebactam (98.7% susceptible) showed similar activity to ceftazidime-avibactam (99.5% susceptible) and higher than ceftolozane-tazobactam (86.9% susceptible). Imipenem-relebactam was inactive against 1.3% (10/747) isolates, all of them due to carbapenemase production (9 K. pneumoniae and 1 E. cloacae). Imipenem-relebactam was active against 100% of extended-spectrum ß-lactamase (ESBL)-E. coli and ESBL-Klebsiella spp. isolates and 80.4% of carbapenemase-Klebsiella spp. producers. Carbapenemase genes were confirmed by WGS in 41 Klebsiella spp.: OXA-48 (20/41), KPC-3 (14/41), OXA-181 (4/41), NDM-1 (1/41), OXA-48 + VIM-2 (1/41), and KPC-3 + VIM-2 (1/41). In Klebsiella spp. isolates, relebactam restored imipenem susceptibility in all KPC-3 producers, and resistant isolates (7/41) were mostly OXA-48 + CTX-M-15-K. pneumoniae high-risk clones (7/9). Intercountry differences were detected as follows: OXA-48 (17/21) was dominant in Spain, unlike KPC-3 (14/15) in Portugal. Imipenem-relebactam was 100% active against CTX-M-15-ST131-H30Rx-E. coli high-risk clone, predominant in both countries. Our results depict the potential role of imipenem-relebactam in ICU patients with cIAIs, cUTIs, and LRTIs due to wild-type ESBL- and carbapenemase-producing Enterobacterales, particularly KPC producers. IMPORTANCE We comparatively evaluate the in vitro activity of a drug combination consisting of a carbapenem (imipenem) and a novel inhibitor of beta-lactamases (relebactam), a mechanism that destroys beta-lactam antibiotics. We assess the activity against a collection of Enterobacterales clinical isolates recovered from difficult-to-treat infections in patients admitted to different intensive care units in Portugal and Spain. Imipenem-relebactam shows excellent activity in avoiding common resistance mechanisms in this setting, such as extended-spectrum beta-lactamases and carbapenemases widely distributed, including KPCs. We show few resistant isolates (<2%). Molecular characterization by whole-genome sequencing shows that most of the resistant isolates produced specific carbapenemase, such as OXA-48 or metalo-betalactamases. Our study updates the activity of imipenem-relebactam in light of current epidemiology in a hospital setting in which the use of this combination is needed due to the presence of infections due to multidrug-resistant isolates.

Epidemiology of Clostridioides difficile Infection in Portugal: A Retrospective, Observational Study of Hospitalized Patients.

INTRODUCTION: Clostridioides difficile is the main cause of healthcare-associated diarrhea in Europe and North America. The aim of this study was to characterize the epidemiology and clinical burden of Clostridioides difficile infection among hospitalized patients in Portugal. MATERIAL AND METHODS: Retrospective study conducted in six public hospital centers in Portugal. All primary Clostridioides difficile infection episodes and related recurrences occurring in 2017, as well as episodes developing two to eight weeks after the last episode diagnosed in that year, were documented. The National Reference Laboratory (National Institute of Health Dr. Ricardo Jorge) provided national surveillance data on Clostridioides difficile infection. RESULTS: A total of 385 inpatients with at least one primary episode diagnosed in 2017 were included. Most patients were aged over 70 years-old (73.2%). The included patients developed 451 episodes during the observation period. Approximately 44% of primary episodes were community-associated. Most episodes (94.9%) occurred in patients with one or more risk factors, with recent antibiotic exposure being particularly common (86.0%). All-cause in-hospital mortality was 19.5%, being significantly higher in patients aged over 65 years-old versus those aged 18 to 64 years-old (22.4% vs 7.8%, respectively). Over 50 different ribotypes were observed among 206 Clostridioides difficile strains received by the National Reference Laboratory. CONCLUSION: In Portugal, hospitalized patients with Clostridioides difficile infection are mostly older patients presenting risk factors for the development of this infection, particularly recent antibiotic exposure. Mortality is disproportionately high among the older population. Community-associated Clostridioides difficile infection is common among inpatients with this infection.

Introdução: Clostridioides difficile é a principal causa de diarreia nosocomial na Europa e América do Norte. Este estudo teve como objetivo caracterizar a epidemiologia e o impacto clínico da infeção por Clostridioides difficile em doentes hospitalizados em Portugal. Material e Métodos: Estudo retrospetivo conduzido em seis centros hospitalares públicos de Portugal. Foram documentados todos os episódios primários de infeção por Clostridioides difficile ocorridos em 2017 e consequentes recorrências, bem como os episódios que ocorreram entre duas a oito semanas após o último episódio diagnosticado neste ano. Os dados de vigilância nacional de infeção por Clostridioides difficile foram fornecidos pelo laboratório nacional de referência (Instituto Nacional de Saúde Doutor Ricardo Jorge). Resultados: Foram incluídos 385 doentes hospitalizados com pelo menos um episódio primário diagnosticado em 2017. A maioria dos doentes tinha idade igual ou superior a 70 anos (73,2%). Os doentes incluídos tiveram 451 episódios durante o período de observação. Aproximadamente 44% dos episódios primários eram episódios de infeção por Clostridioides difficile adquirida na comunidade. A maioria dos episódios (91,8%) ocorreu em doentes com um ou mais fatores de risco, sendo a exposição recente a antibióticos particularmente comum (86,0%). A mortalidade hospitalar por todas as causas foi de 19,5%, sendo significativamente superior em doentes com idade igual ou superior a 65 anos comparativamente a doentes com idade entre 18 e 64 anos (22,4% versus 7,8%, respetivamente). Mais de 50 ribotipos diferentes foram detetados entre as 206 estirpes de Clostridioides difficile recebidas pelo laboratório nacional de referência. Conclusão: Em Portugal, doentes hospitalizados com infeção por Clostridioides difficile são, na sua maioria, doentes idosos com fatores de risco para o seu desenvolvimento, particularmente exposição recente a antibióticos. A mortalidade é desproporcionalmente elevada na população idosa. Episódios associados à comunidade são comuns em doentes hospitalizados com esta infeção.

Confronting Ceftolozane-Tazobactam Susceptibility in Multidrug-Resistant Enterobacterales Isolates and Whole-Genome Sequencing Results (STEP Study).

Ceftolozane-tazobactam (C/T) is frequently used for infections caused by multidrug-resistant (MDR)-Enterobacterales isolates. Whole-genome sequencing (WGS, Illumina-Hiseq 4000/NovaSeq 6000, OGC, UK) was used to study the population structure, the resistome and the virulome of C/T-susceptible and -resistant MDR Escherichia spp. (n=30) and Klebsiella spp. (n=78) isolates, recovered from lower respiratory, intra-abdominal and urinary tract infections of ICU patients from 11 Portuguese Hospitals (STEP study, 2017-2018). Minimum inhibitory concentrations (MICs) were determined (ISO-broth microdilution, breakpoints EUCAST-2020). In Escherichia spp., a weak concordance between the phenotypic and the WGS method (P=0.051) was observed in the carbapenemase detection (3/30) [blaVIM-2 (2/3), blaKPC-3 (1/3)]; VIM-2-Escherichia coli isolates were C/T-susceptible and only the KPC-3-Escherichia marmotae producer showed C/T-resistance. Overall, CTX-M-15-E. coli-ST131-O25:H4-H30-Rx (11/30) was the most frequent subclone, followed by CTX-M-27-E. coli-ST131-O25:H4-H30 (4/4). Moreover, a wide resistome and virulome were detected in all E. coli isolates. Among Klebsiella spp. isolates [K. pneumoniae (67/78), K. aerogenes (7/78), K. oxytoca (2/78), K. variicola (2/78)], concordance (P<0.001) was observed between the phenotypic and the genomic carbapenemase detection (21/78) [blaKPC-3 (14/21), blaOXA-48 (3/21), blaOXA-181 (3/21)]. A high correlation between C/T-resistance and carbapenemase detection was established (P<0.05). Overall, a high clonal diversity was observed, mainly in KPC-3-producing K. pneumoniae isolates. An extensive resistome was detected in Klebsiella spp. isolates, whereas virulence determinants were mostly identified in carbapenemase producers (P<0.001). WGS is a powerful tool for typing characterization and microbiological study of MDR-Enterobacterales pathogens. Furthermore, carbapenemase genes are associated with C/T-resistance in Klebsiella spp., but other mechanisms might also be involved.

Distinct epidemiology and resistance mechanisms affecting ceftolozane/tazobactam in Pseudomonas aeruginosa isolates recovered from ICU patients in Spain and Portugal depicted by WGS.

OBJECTIVES: To analyse the epidemiology, the resistome and the virulome of ceftolozane/tazobactam-susceptible or -resistant Pseudomonas aeruginosa clinical isolates recovered from surveillance studies in Portugal (STEP, 2017-18) and Spain (SUPERIOR, 2016-17). METHODS: P. aeruginosa isolates were recovered from intra-abdominal, urinary tract and lower respiratory tract infections in ICU patients admitted to 11 Portuguese and 8 Spanish hospitals. MICs were determined (ISO-standard broth microdilution, EUCAST 2020 breakpoints). A subset of 28 ceftolozane/tazobactam-resistant P. aeruginosa isolates were analysed and compared with 28 ceftolozane/tazobactam-susceptible P. aeruginosa strains by WGS. RESULTS: Clonal complex (CC) 235 (27%) and CC175 (18%) were the most frequent, followed by CC244 (13%), CC348 (9%), CC253 (5%) and CC309 (5%). Inter-hospital clonal dissemination was observed, limited to a geographical region (CC235, CC244, CC348 and CC253 in Portugal and CC175 and CC309 in Spain). Carbapenemases were detected in 25 isolates (45%): GES-13 (13/25); VIM type (10/25) [VIM-2 (4/10), VIM-20 (3/10), VIM-1 (2/10) and VIM-36 (1/10)]; and KPC-3 (2/25). GES-13-CC235 (13/15) and VIM type-CC175 (5/10) associations were observed. Interestingly, KPC-3 and VIM-36 producers showed ceftolozane/tazobactam-susceptible phenotypes. However, ceftolozane/tazobactam resistance was significantly associated with GES-13 and VIM-type carbapenemase production. Six non-carbapenemase producers also displayed ceftolozane/tazobactam resistance, three of them showing known ceftolozane/tazobactam resistance-associated mutations in the PBP3 gene, ftsI (R504C and F533L). Overall, an extensive virulome was identified in all P. aeruginosa isolates, particularly in carbapenemase-producing strains. CONCLUSIONS: GES-13-CC235 and VIM type-CC175 were the most frequent MDR/XDR P. aeruginosa clones causing infections in Portuguese and Spanish ICU patients, respectively. Ceftolozane/tazobactam resistance was mainly due to carbapenemase production, although mutations in PBP-encoding genes may additionally be involved.

In vitro activity of ceftolozane-tazobactam against Enterobacterales and Pseudomonas aeruginosa causing urinary, intra-abdominal and lower respiratory tract infections in intensive care units in Portugal: The STEP multicenter study.

The STEP surveillance study was designed to increase knowledge about distribution of multidrug-resistant (MDR) Enterobacterales and Pseudomonas aeruginosa in Portugal, focusing on the intensive care unit (ICU). Antimicrobial susceptibility of common agents was also evaluated and compared with that of one of the latest therapeutic introductions, ceftolozane-tazobactam (C/T). Clinical isolates of Enterobacterales (n=426) and P. aeruginosa (n=396) from patients admitted in Portuguese ICUs were included. Activity of C/T and comparators was investigated using standard broth microdilution. Isolates were recovered from urinary tract (UTI, 36.9%), intra-abdominal (IAI, 24.2%) and lower respiratory tract (LRTI, 38.9%) infections. In P. aeruginosa, overall distribution of MDR/extremely-drug resistant (XDR)/pan-drug resistant (PDR) isolates accounted for 21.2%, 23.2% and 0.8%, respectively. C/T was the most potent agent tested against P. aeruginosa and MDR/XDR/PDR phenotypes. In Escherichia coli, extended-spectrum beta-lactamases (ESBL) and carbapenemase (CP) phenotypes accounted for 16.6% and 1.7%, respectively, whereas in Klebsiella spp., ESBL and CP-phenotypes represented 28.5% and 17.9%, respectively. Overall, susceptibility of C/T against Enterobacterales was 86.9%. C/T was the least affected agent in E. coli (99.4% susceptibility), whereas its activity was moderate in Klebsiella spp. (71.5%) and Enterobacter spp. (70.4%), due in part to a high rate of ESBL and CP-phenotypes. In Enterobacterales, blaKPC was the most prevalent CP gene (63.0%), followed by blaOXA-48 (33.3%) and blaVIM (3.7%). These microbiological results reinforce C/T as a therapeutic option in ICU patients with UTI, IAI or LRTI due to P. aeruginosa or Enterobacterales isolates, but not for CP producers.

Longitudinal analysis of the in vitro activity of ceftazidime/avibactam versus Enterobacteriaceae, 2012-2016.

OBJECTIVES: The in vitro activities of ceftazidime/avibactam and comparator antimicrobial agents were analysed against 59 828 Enterobacteriaceae isolates collected by 190 centres from all global regions except North America from 2012-2016 as part of the International Network for Optimal Resistance Monitoring (INFORM) global surveillance programme. METHODS: Antimicrobial susceptibility testing was performed using Clinical and Laboratory Standards Institute broth microdilution panels at a central reference laboratory, except for isolates collected in China that were tested using frozen, dehydrated broth microdilution panels at a central laboratory in China. The presence of extended-spectrum ß-lactamases (ESBLs) was confirmed by multiplex PCR assays. RESULTS: Ceftazidime/avibactam was the most active agent against all Enterobacteriaceae (MIC90, ≤1mg/L, ≥98.4% susceptibility). High rates of susceptibility (>88%) were observed amongst Citrobacter freundii, Citrobacter spp., Enterobacter spp., Escherichia coli, Klebsiella pneumoniae and Klebsiella oxytoca to colistin, meropenem, amikacin and tigecycline. Ceftazidime/avibactam showed consistent in vitro activity against ESBL-positive isolates of E. coli (n=5674; MIC90, 0.5mg/L, 99.5% susceptible), K. pneumoniae (n=7097; MIC90, 2mg/L, 97.0% susceptible) and K. oxytoca (n = 565; MIC90, 1mg/L, 96.8% susceptible). Isolates identified as metallo-ß-lactamase-positive (n=242) were not susceptible to ceftazidime/avibactam but were susceptible to tigecycline (76.9%) and colistin (n=194 isolates tested; 92.8%). CONCLUSIONS: Clinical Enterobacteriaceae isolates, including ESBL-positive phenotypes, collected globally (excluding North America) from 2012-2016 were highly susceptible to ceftazidime/avibactam, suggesting it is a useful agent for serious infections caused by multidrug-resistant organisms belonging to the family Enterobacteriaceae when therapeutic options are limited.

Evaluation of vancomycin MIC creep in Staphylococcus aureus.

OBJECTIVES: Vancomycin is the primary treatment for methicillin-resistant Staphylococcus aureus (MRSA). However, an increasing proportion of MRSA isolates with high minimum inhibitory concentrations (MICs) within the susceptible range (vancomycin 'MIC creep') is being observed. The aim of this study was to assess the vancomycin MIC distribution for S. aureus isolates over a period of 4 years in Centro Hospitalar Baixo Vouga (Aveiro, Portugal) and to identify differences in vancomycin MIC determined by different susceptibility testing methods. METHODS: For each S. aureus isolate, the vancomycin MIC was assayed by the VITEK®2 automated system and the broth microdilution testing method. RESULTS: The results showed significant differences in vancomycin MIC by different methods (P=0.021, sign test) and did not suggest the presence of vancomycin MIC creep during the study period. CONCLUSIONS: Vancomycin MIC creep is a regional problem, therefore it can only be assessed through the evaluation of local susceptibility profiles, and antibiogram based on real MIC assay should be an essential element in local MRSA infection clinical management.

mcr-1 and blaKPC-3 in Escherichia coli Sequence Type 744 after Meropenem and Colistin Therapy, Portugal.

Escherichia coli Ec36 was recovered from a patient in Portugal after treatment with meropenem and colistin. Besides an IncF plasmid with Tn1441d-blaKPC-3, already reported in clinical strains in this country, E. coli Ec36 co-harbored an IncX4::mcr-1 gene. Results highlight emerging co-resistance to carbapenems and polymyxins after therapy with drugs from both classes.

Proteomic profile of susceptible and multidrug-resistant clinical isolates of Escherichia coli and Klebsiella pneumoniae using label-free and immunoproteomic strategies.

Infectious diseases caused by multidrug-resistant (MDR) Enterobacteriaceae have exponentially increased in the past decade, and are a major concern in hospitals. In the first part of the work, we compared the proteome profile of MDR and susceptible clinical isolates of Escherichia coli and Klebsiella pneumoniae in order to identify possible biological processes associated with drug resistance and susceptible phenotypes, using a label-free approach. In the second part, we used an immunoproteomics approach to identify immunoreactive proteins in the same isolates. A total of 388 and 377 proteins were identified in MDR and susceptible E. coli, respectively, evidencing that biological processes related to translation are upregulated in E. coli MDR, while there is an upregulation of processes related to catalytic activity in K. pneumoniae MDR. Both MDR strains show downregulation of processes related to amino acid activation and tRNA amino-acylation. Our data also suggest that MDR strains have higher immunoreactivity than the susceptible strains. The application of high-throughput mass spectrometry (MS) and bioinformatics to the study of modulation of biological processes might shed light on the characterization of multidrug resistance in bacteria.

Genetically unrelated multidrug- and carbapenem-resistant Citrobacter freundii detected in outpatients admitted to a Portuguese hospital.

OBJECTIVES: Non-clonal, carbapenem- and multidrug-resistant Citrobacter freundii isolates were collected from unrelated outpatients admitted to a Portuguese hospital emergency department. One patient lived in a nursing home and was never hospitalised, whereas the other patient was repeatedly hospitalised in this hospital. The aim of this study was to unveil the molecular mechanisms associated with the carbapenem resistance of these isolates and to assess its potential dissemination. METHODS: Isolate identification was performed by VITEK®2 and was confirmed by 16S rDNA sequencing. The clonal relationship of the isolates was evaluated by repetitive element palindromic PCR (rep-PCR). Antibiotic susceptibility was determined using the automatic VITEK®2 AES system and disk diffusion assay. ß-Lactamases, porins and mobile genetic elements were characterised by PCR and sequencing. Pulsed-field gel electrophoresis (PFGE) and Southern blot hybridisation were used to determine the genetic location of integrons, and their transferability was tested by conjugation. RESULTS: No genetic relatedness was found, suggesting different origins of the isolates. In isolate Cf254 a VIM-2 carbapenemase integrated in In58 was detected, located in a high-frequency conjugative IncL/M plasmid that also carried CTX-M-15 and CMY-39 genes. VIM-1 in isolate Cf872 was chromosomal. This is the first description in Portugal of VIM carbapenemases in C. freundii. Loss/alteration of porins was also detected. CONCLUSIONS: Emergence of carbapenem-resistant Enterobacteria is not confined to the nosocomial environment. Community-acquired strains appear to be in circulation between inpatients and outpatients, spreading carbapenem resistance genes by horizontal gene transfer.

Physicians' attitudes and knowledge concerning antibiotic prescription and resistance: questionnaire development and reliability.

BACKGROUND: Understanding physicians' antibiotic-prescribing behaviour is fundamental when it comes to improving antibiotic use and tackling the growing rates of antimicrobial resistance. The aim of the study was to develop and validate--in terms of face validity, content validity and reliability--an instrument designed to assess the attitudes and knowledge underlying physician antibiotic prescribing. METHODS: The questionnaire development and validation process comprised two different steps, namely: (1) content and face validation, which included a literature review and validation both by physicians and by Portuguese language and clinical psychology experts; and (2) reliability analysis, using the test-retest method, to assess the questionnaire's internal consistency (Cronbach's alpha) and reproducibility (intraclass correlation coefficient--ICC). The questionnaire includes 17 items assessing attitudes and knowledge about antibiotic prescribing and resistances and 9 items evaluating the importance of different sources of knowledge. The study was conducted in the catchment area covered by Portugal's Northern Regional Health Administration and used a convenience sample of 61 primary-care and 50 hospital-care physicians. RESULTS: Response rate was 64% (49% to retest) for primary-care physicians and 66% (60% to retest) for hospital-care physicians. Content validity resulted in 9 changes to professional concepts. Face validity assessment resulted in 19 changes to linguistic and interpretative terms. In the case of the reliability analysis, the ICC values indicated a minimum of fair to good reproducibility (ICC > 0.4), and the Cronbach alpha values were satisfactory (α > 0.70). CONCLUSIONS: The questionnaire developed is valid--in terms of face validity, content validity and reliability--for assessing physicians' attitudes to and knowledge of antibiotic prescribing and resistance, in both hospital and primary-care settings, and could be a very useful tool for characterising physicians' antibiotic-prescribing behaviour.

Methicillin-resistant Staphylococcus aureus carrying the new mecC gene--a meta-analysis.

In 2011, a new mecA gene homolog, named mecC gene, was found in isolates from both humans and animals. The discovery of methicillin-resistant Staphylococcus aureus (MRSA) carrying the mecC gene has caused speculations about the origin, epidemiology, and impact of these isolates. The objective of this work is to perform a meta-analysis on the prevalence of mecC MRSA, based on previously published results. Meta-analysis showed that the overall pooled prevalence is 0.009% (95% confidence interval=0.05-0.013) and that there was evidence of heterogeneity (P<0.01, I(2)=97%). In conclusion, the very low reported prevalence provides an important baseline to monitor the epidemiology of this emerging form of MRSA.

Clinical characteristics of patients with community-acquired complicated intra-abdominal infections: a prospective, multicentre, observational study.

In this prospective, observational, multicentre study using data from five countries (Columbia, The Philippines, Portugal, Taiwan and Thailand), the clinical impact of extended-spectrum ß-lactamase (ESBL)-producing organisms on hospitalised patients with community-acquired complicated intra-abdominal infections (CA-cIAIs) was compared with that of non-ESBL-producing organisms during the period April 2010 to December 2011. Adult patients (aged ≥18 years) requiring surgery or percutaneous drainage were enrolled and were followed during the first hospitalisation course. An unadjusted statistical comparison of risk factors for ESBL-positive and ESBL-negative patients was performed. Multivariate regression analyses were performed to assess whether length of stay (LOS) in hospital, clinical cure rate and some important clinical characteristics were associated with ESBL positivity. During the study period, a total of 105 adult patients from five countries were enrolled, of whom 17 (16.2%) had CA-cIAI due to ESBL-positive organisms and 88 (83.8%) had CA-cIAI due to ESBL-negative organisms. Escherichia coli was isolated in 73.3% of all samples. Infections were cured in 8 (47.1%) of the patients with CA-cIAI due to ESBL-positive organisms and in 59 (67.0%) of the patients with CA-cIAI due to ESBL-negative organisms (P=0.285). The median LOS was 11.6 days for patients with infections due to ESBL-negative organisms and 17.6 days for patients with infections due to ESBL-positive organisms (P=0.011). Multivariate logistic regression analysis revealed that pre-existing co-morbidities, but not ESBL positivity, were adversely associated with clinical cure of CA-cIAIs. In contrast, duration of hospitalisation was longer for patients with CA-cIAI due to ESBL-positive organisms.

Characterization of the toxicological effects of aminocarb on rats: hematological, biochemical, and histological analyses.

Aminocarb is a widely applied carbamate insecticide with action of controlling pests such as Lepidoptera and Coleoptera. In this study, subchronic effects on Wistar rats were investigated using hematological, biochemical, and histological techniques. Rats were exposed orally at sublethal levels of 10, 20, or 40 mg/kg body weight (groups A, B, and C, respectively) for 14 d. Hematological results revealed no statistical differences after 1 d of exposure but significant reduction in white blood cells detected after 7 d of exposure in group C, as well as, in all treated groups after 14 d of exposure. Biochemical data showed a decrease of acetylcholinesterase activity in all groups after 1 d of exposure with a return to normal after 7 and 14 d. Significant increase in alkaline phosphatase activity of rats exposed to aminocarb was noted after 7 d of treatment. The levels of triglycerides were also significantly decreased. The present investigation also showed a significant increase in content of serum urea and creatinine in animals from group A (14 d), and from groups B and C (7 and 14 d). Histological results demonstrated hemorrhagic focus on hepatic and renal parenchyma in all exposed groups. Taken together, the attained results were dose dependent and indicated adverse effects of aminocarb on hepatic and renal functions, as well as on immune responsiveness at sublethal tested doses.

Subacute effects of the thiodicarb pesticide on target organs of male Wistar rats: biochemical, histological, and flow cytometry studies.

Thiodicarb, a carbamate pesticide widely used on crops, may pose several environmental and health concerns. This study aimed to explore its toxicological profile on male rats using hematological, biochemical, histopathological, and flow cytometry markers. Exposed animals were dosed daily at 10, 20, or 40 mg/kg/body weight (group A, B, and C, respectively) during 30 d. No significant changes were observed in hematological parameters among all groups. After 10 d, a decrease of total cholesterol levels was noted in rats exposed to 40 mg/kg. Aspartate aminotransferase (AST) activity increased (group A at 20 d; groups A and B at 30 d) and alkaline phosphatase (ALP) (group B at 30 d) activity significantly reduced. At 30 d a decrease of some of the other evaluated parameters was observed with total cholesterol and urea levels in group A as well as total protein and creatinine levels in groups A and B. Histological results demonstrated multi-organ dose-related damage in thiodicarb-exposed animals, evidenced as hemorrhagic and diffuse vacuolation in hepatic tissue; renal histology showed disorganized glomeruli and tubular cell degeneration; spleen was ruptured with white pulp and clusters of iron deposits within red pulp; significant cellular loss was noted at the cortex of thymus; and degenerative changes were observed within testis. The histopathologic alterations were most prominent in the high-dose group. Concerning flow cytometry studies, an increase of lymphocyte number, especially T lymphocytes, was seen in blood samples from animals exposed to the highest dose. Taken together, these results indicate marked systemic organ toxicity in rats after subacute exposure to thiodicarb.

First description of Klebsiella pneumoniae clinical isolates carrying both qnrA and qnrB genes in Portugal.

In the present study, 21 multidrug-resistant Klebsiella pneumoniae isolates were recovered from patients hospitalised in the Intensive Care Unit of Hospital Infante D. Pedro in Aveiro, Portugal. Fifteen isolates carried qnr genes. Four strains harboured the quinolone resistance genes qnrA and qnrB, both located on a large plasmid in two strains (KP4 and KP10) and on different plasmids in two strains (KP5 and KP6). These findings indicate an extremely high prevalence of qnr genes associated with various mobile elements such as ISCR1 and class 1 integrons.

Carriage of qnrA1 and qnrB2, blaCTX-M15, and complex class 1 integron in a clinical multiresistant Citrobacter freundii isolate.

A multiresistant Citrobacter freundii strain was recovered from a catheter from a patient hospitalized in Aveiro, Portugal. This strain harbored quinolone resistance genes, qnrA1 and qnrB2, both in a large plasmid.

[Streptococcal pharyngitis: clinical suspicion versus diagnosis]. / Amigdalite estreptocócica: presunção clínica versus diagnóstico.

INTRODUCTION: Pharyngitis is a very prevalent illness in the ambulatory care setting. Its diagnosis is a challenge, especially in the differentiation between the viric and streptococcal causes. MATERIALS AND METHODS: A formulary was made to register the clinical and laboratory data; a throat swab for culture was obtained from all the children who presented to the emergency department with sore throat and/or signs of pharyngitis/tonsillitis, for a period of three months (15th of April to 15th of July of 2006). The signs and symptoms, prescribed antibiotherapy and frequency of false diagnostics were evaluated and the clinical suspicion compared with the diagnosis by culture. RESULTS: 158 children were evaluated, with a median age of four years, with a male predominance (56%). The period that showed the greatest number of cases was the first fifteen days of May. Forty-three percent of the cultures were positive for Streptococcus pyogenes. The more frequent signs and symptoms in pharyngitis were pharyngeal erythema (98%), fever (86%) and sore throat (78%). A significative statistical difference was found for cough, scarlatiniform rash, tonsillar exudate, palatal petechiae and tonsillar swelling. Of the signs and symptoms studied, only three of them presented a positive predictive value superior to 50%: scarlatiniform rash (85%), palatal petechiae (63%) and cough (57%). The presence of tonsillar exudate had a positive predictive value for non-streptococcal pharyngitis of 70%. Fifty-three percent of the doctors considered streptococcal pharyngitis highly probable, and from this, 56% had a positive culture for Streptococcus. Those who considered a low probability, the culture was positive in 28%. There were 37% of false diagnosis. DISCUSSION: The distinction between streptococcal pharyngitis and non-streptococcal pharyngitis is not always correct when based on clinical characteristics. The use of diagnostic tests is important in order to avoid unnecessary antibiotherapy as well as to allow the correct use in the positive cases.