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Introduction: Despite being implicated in a wide spectrum of community- and healthcare-acquired infections, anaerobes have not yet been incorporated into systematic surveillance programs in Europe., Methods: We conducted a multicentre retrospective observational study analysing all anaerobic strains isolated from blood cultures in 44 European Hospital Centres over a 4-year period (2020-2023). Diagnostic approach, epidemiology, and antimicrobial susceptibility according to EUCAST v. 15.0 were investigated., Results: Our study included 14,527 anaerobes, most of which were Gram-positive (45%) or Gram-negative (40%) bacilli. MALDI-TOF coupled to mass spectrometry was the most widely used tool for species identification (98%). Antimicrobial susceptibility testing was performed in the vast majority of centres, using mostly gradient diffusion strip (77%) and disk diffusion (45%) methods according to EUCAST guidelines (v. 15.0). The most prevalent species were Cutibacterium acnes (18.7%), Bacteroides fragilis (16.3%), Clostridium perfringens (5.3%), Bacteroides thetaiotaomicron (4.2%), Fusobacterium nucleatum (3.5%), and Parvimonas micra (3.4%). C. acnes showed high resistance to benzylpenicillin (18%), clindamycin (39%), and imipenem (19% and 13% by MIC methods and disk diffusion, respectively). B. fragilis showed high resistance to amoxicillin/clavulanate (24%), piperacillin/tazobactam (22% and 14% by MIC methods and disk diffusion, respectively), clindamycin (22% by both MIC methods and disk diffusion), meropenem (13%), and metronidazole (10%, only by disk diffusion). A similar resistance pattern was observed in B. thetaiotaomicron, Bacteroides ovatus, and Parabacteroides distasonis. C. perfringens showed high resistance to clindamycin (69% and 45% by MIC methods and disk diffusion, respectively), while benzylpenicillin and metronidazole maintained over 90% activity. F. nucleatum showed high resistance to benzylpenicillin (11%), while Fusobacterium necrophorum showed alarming rates of resistance to clindamycin (12%), meropenem (16%) and metronidazole (11%)., Conclusions: This study presented an up-to-date analysis of the diagnostics and epidemiology of anaerobic bacteria in Europe, providing insights for future comparative analyses and the development of antimicrobial diagnostic and management strategies, as well as the optimization of current antibiotic treatments., Competing Interests: Competing Interests None., (Copyright © 2025. Published by Elsevier Ltd.)

Carbapenem-resistant Klebsiella pneumoniae (CR-KP) are a public health concern, causing infections with a high mortality rate, limited therapeutic options and challenging infection control strategies. In Portugal, the CR-KP rate has increased sharply, but the factors associated with this increase are poorly explored. In order to address this question, phylogenetic and resistome analysis were used to compare the draft genomes of 200 CR-KP isolates collected in 2017-2019 from five hospitals in the Lisbon region, Portugal. Most CR-KP belonged to sequence type (ST) 13 (29%), ST17 (15%), ST348 (13%), ST231 (12%) and ST147 (7%). Carbapenem resistance was conferred mostly by the presence of KPC-3 (74%) or OXA-181 (18%), which were associated with IncF/IncN and IncX plasmids, respectively. Almost all isolates were multi-drug resistant, harbouring resistance determinants to aminoglycosides, beta-lactams, trimethoprim, fosfomycin, quinolones and sulphonamides. In addition, 11% of isolates were resistant to colistin. Colonizing and infecting isolates were highly related, and most colonized patients (89%) reported a previous hospitalization. Moreover, among the 171 events of cross-dissemination identified by core genome multi-locus sequence typing data analysis (fewer than five allelic differences), 41 occurred between different hospitals and 130 occurred within the same hospital. The results suggest that CR-KP dissemination in the Lisbon region results from acquisition of carbapenemases in mobile genetic elements, influx of CR-KP into the hospitals by colonized ambulatory patients, and transmission of CR-KP within and between hospitals. Prudent use of carbapenems, patient screening at hospital entry, and improvement of infection control are needed to decrease the burden of CR-KP infection in Portugal., (Copyright © 2024 The Healthcare Infection Society. Published by Elsevier Ltd. All rights reserved.)

Crystalline inclusions in lymphocytes are unusual in chronic lymphocytic leukemia. They represent crystallized immunoglobulin (most frequently IgM-lambda) and their recognition facilitates the diagnosis of a lymphoproliferative disorder., (Copyright © 2022 Elsevier Masson SAS. All rights reserved.)

Competing Interests: Conflicts of interest None.

Objectives: To describe our experience with a coronavirus disease 2019 (COVID-19) outbreak within a large rheumatology department early in the pandemic. Methods: Symptomatic and asymptomatic healthcare workers (HCWs) had a naso-oropharyngeal swab for detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and were followed clinically. Reverse transcription polymerase-chain reaction (RT-PCR) was repeated to document cure, and serological response was assessed. Patients with risk contacts within the department in the 14 days preceding the outbreak were screened for COVID-19 symptoms. Results: 14/34 HCWs (41%; 40 ± 14 years, 71% female) tested positive for SARS-CoV-2, and 11/34 (32%) developed symptoms but were RT-PCR-negative. Half of RT-PCR-positive HCWs did not report fever, cough, or dyspnea before testing, which were absent in 3/14 cases (21%). Mild disease prevailed (79%), but 3 HCWs had moderate disease requiring further assessment, which excluded severe complications. Nevertheless, symptom duration (28 ± 18 days), viral shedding (31 ± 10 days post-symptom onset, range 15-51), and work absence (29 ± 28 days) were prolonged. 13/14 (93%) of RT-PCR-positive and none of the RT-PCR-negative HCWs had a positive humoral response Higher IgG indexes were observed in individuals over 50 years of age (14.5 ± 7.7 vs. 5.0 ± 4.4, p = 0.012). Of 617 rheumatic patients, 8 (1.3%) developed COVID-19 symptoms (1/8 hospitalization, 8/8 complete recovery), following a consultation/procedure with an asymptomatic (7/8) or mildly symptomatic (1/8) HCW. Conclusions: A COVID-19 outbreak can occur among HCWs and rheumatic patients, swiftly spreading over the presymptomatic stage. Mild disease without typical symptoms should be recognized and may evolve with delayed viral shedding, prolonged recovery, and adequate immune response in most individuals., (Copyright © 2020 Romão, Oliveira-Ramos, Cruz-Machado, Martins, Barreira, Silva-Dinis, Mendonça-Galaio, Proença, Cristino, Sacadura-Leite, Khmelinskii, Romeu, Fonseca and the CHULN Rheumatology Department.)

Introduction: Advances in molecular diagnosis have made it possible to detect previously unknown viral agents as causative agents of lower respiratory tract infections (LRTI). The frequency and relevance of viral coinfections is still debatable., Objective: compare clinical presentation and severity between single virus infection and viral coinfection in children admitted for LRTI., Methods: A 3-year period observational study (2012-2015) included children younger than two years admitted for LRTI. Viral identification was performed using PCR technique for 16 viruses. Clinical data and use of health resources was gathered during hospital stay using a standard collection form and we compared single virus infection and viral coinfections., Results: The study included 524 samples (451 patients); 448 (85,5%) had at least one virus identified. Viral coinfections were found in 159 (35,5%). RSV and HRV were the most commonly identified virus; bronchiolitis and pneumonia the most frequent diagnosis. Patients with viral coinfections were older, attended day-care centers, had previous recurrent wheezing more frequently and were more symptomatic at admission. These patients did not have more complementary exams performed but were prescribed medications more often. Viral coinfection group did not show longer length of hospital stay and oxygen need, more need for ICU nor ventilatory support., Discussion: Our study showed a significant proportion of viral coinfections in young infants admitted with LRTI and confirmed previous data showing that prescription was more frequent in inpatients with viral coinfections, without an association with worst clinical outcome., (Copyright © 2017 Asociación Española de Pediatría. Publicado por Elsevier España, S.L.U. All rights reserved.)

Listeria monocytogenes isolates (n = 81) recovered from ready-to-eat meat-based food products (RTEMP) collected in industrial processing plants and retail establishments were genetically characterized for comparison with those from human clinical cases of listeriosis (n = 49). The aim was to assess RTEMP as a possible food source for human infection. L. monocytogenes was detected in 12.5% of the RTEMP samples, and in some cases, counts were above the European food safety criteria. All isolates were assessed by multiplex PCR for serogroup determination and detection of virulence-associated genes inlA, inlB, inlC, inlJ, plcA, hlyA, actA, and iap. Serogroups IIb and IVb dominated in RTEMP and human isolates, and all were positive for the assessed virulence genes. Antibiotic susceptibility testing by the disk diffusion method revealed a low level of resistance among the isolates. Pulsed-field gel electrophoresis (PFGE) of L. monocytogenes isolates, using restriction enzymes ApaI and AscI, revealed genetic variability and differentiated the isolates in five clusters. Although some pulsed-field gel electrophoresis profiles of particular RTEMP and human isolates seemed to be highly related, exhibiting more than 90% similarity, which suggests a possible common source, in most cases the strains were not genetically or temporally matched. The close genetic relatedness of RTEMP and human listeriosis strains stressed the importance of preventive measure implementation throughout the food chain.

Diabetes mellitus is a major chronic disease that continues to increase significantly. One of the most important and costly complications of diabetes is foot ulceration that may be colonized by pathogenic and antimicrobial resistant bacteria, which may express several virulence factors that could impair treatment success. These bacterial communities can be organized in polymicrobial biofilms, which may be responsible for diabetic foot ulcer (DFU) chronicity. We evaluated the influence of polymicrobial communities in the ability of DFU isolates to produce biofilm, using a microtiter plate assay and a multiplex fluorescent in situ hybridization, at three time points (24, 48, 72 h), after evaluating biofilm formation by 95 DFU isolates belonging to several bacterial genera (Staphylococcus, Corynebacterium, Enterococcus, Pseudomonas and Acinetobacter). All isolates were biofilm-positive at 24 h, and the amount of biofilm produced increased with incubation time. Pseudomonas presented the higher biofilm production, followed by Corynebacterium, Acinetobacter, Staphylococcus and Enterococcus. Significant differences were found in biofilm formation between the three time points. Polymicrobial communities produced higher biofilm values than individual species. Pseudomonas + Enterococcus, Acinetobacter + Staphylococcus and Corynebacterium + Staphylococcus produced higher biofilm than the ones formed by E. faecalis + Staphylococcus and E. faecalis + Corynebacterium. Synergy between bacteria present in dual or multispecies biofilms has been described, and this work represents the first report on time course of biofilm formation by polymicrobial communities from DFUs including several species. The biological behavior of different bacterial species in polymicrobial biofilms has important clinical implications for the successful treatment of these infections.

Background: Cultures for urinary tract infections (UTI) constitute a large workload in the clinical microbiology laboratory, although up to 80% are usually negative. Several automated methods are available to screen urines for UTI, one being the flow cytometry-based Sysmex((R)) UF-100., Methods: The performance of the UF-100 was evaluated over a 16-month period using urine culture as the reference method., Results: During this period, a total of 5356 urine samples were studied (469 children; 3229 women and 1658 men), of which 706 were culture positive (593 grew Gram negative bacilli). Receiver operating characteristics (ROC) curve analysis showed an area under the curve (AUC) of 0.83 for leukocytes and 0.85 for bacterial count. Applying cut-off values reported in the literature gave sensitivities ranging from 75% to 90%, resulting in 73-174 false negatives (FN). Using a logical combination (leukocytes >or=15x10(6)/L OR bacteria >or=500x10(6)/L) gave a sensitivity of 98%. However, the specificity dropped to 25%, resulting in 15 FN., Conclusions: Screening urine samples for UTI detects a large number of culture positive samples. However, the rather large number of FN observed precludes the use of the UF-100 as a routine screening method to exclude urine samples from culture.

Introduction: Coccidia are important causes of diarrhea that is often indistinguishable from other forms of community-acquired diarrhea. However, the detection of oocysts is often only performed when explicitly requested, as part of the ova and parasite (O&P) examination. Reappraisal and understanding of the accurate staining characteristics of auramine O (AuO), which stains nucleic acids, may permit the inexpensive and reliable identification of coccidian oocysts at routine workup of all fecal samples., Methods: AuO-stained smears were prepared from all stool samples received for stool culture in transport medium (SC) and from concentrated stools received for the ova and parasite (O&P) examination., Results: A total of 3732 samples for stool cultures and 3132 samples for O&P examinations were included. Ninety-one samples (1.3%) from 52 patients yielded Coccidia (45 Cryptosporidium spp and 7 Isospora belli). In seven cases oocysts were only detected in samples sent for stool culture in transport medium. The oocysts showed a typical staining pattern and were easy to recognize. The observation of one smear took only around 30seconds, and the reagents and glass slide for one smear did not exceed US$ 0.03., Conclusions: The screening of all fecal samples with AuO-stained smears is a rapid and inexpensive way to increase the detection of coccidial infections, which in most laboratories can be incorporated into the microscopic workup for mycobacteria.

Between November 2001 and November 2004, 231 Escherichia coli isolates resistant to beta-lactam antibiotics were identified. In 14 isolates, bla(TEM-24) (2 isolates), bla(TEM-52) (5 isolates) and bla(TEM-26) (7 isolates) were identified. In 145 E. coli isolates with the same M13 fingerprinting profile and the same resistance phenotype, the bla(CTX-M-15) gene was found in association with an insertion sequence ISEcp1. The bla(CTX-M-2) gene was identified in one E. coli isolate (290HSM), and in other E. coli isolate (246HSM) the bla(CTX-M-9) gene was contained in a new complex sul1-type class 1 integron (named In60A). This is the first report of three cefotaximases (CTX-M-15, CTX-M-2 and CTX-M-9) in E. coli isolates from a Portuguese hospital.

The conventional BacT/ALERT FA blood cultures supported the ample growth of Mycobacterium tuberculosis in seeding experiments and appeared to perform as reliably as the BACTEC Myco/F-Lytic vials in the recovery of M. tuberculosis from blood in HIV-infected patients. Overall, blood cultures were positive in 39% of patients with tuberculosis.

Klebsiella pneumoniae FFUL 22K was isolated in April 1999 from the urine of an intensive care unit patient in Portugal. The strain showed an extended-spectrum cephalosporin resistance profile. A typical synergistic effect between cefotaxime or cefepime and clavulanic acid was observed. An Escherichia coli transformant displayed a similar resistance phenotype and harbored a ca. 9.4-kb plasmid (p22K9). Cloning experiments revealed that the extended-spectrum beta-lactamase was encoded by bla(GES-1), previously described in class 1 integrons from K. pneumoniae ORI-1 and Pseudomonas aeruginosa Pa695. Further sequence analysis demonstrated that the bla(GES-1) gene cassette was located on a new class 3 integron. The integron was 2863 bp long and consisted of an intI3 integrase gene, an attI3 recombination site, two promoter regions, and two gene cassettes. The IntI3 integrase was 98.8% identical to that of Serratia marcescens AK9373. The bla(GES-1) gene cassette was inserted at the attI3 site. The second gene cassette was the result of a fusion event between bla(OXA-10)-type and aac(6')-Ib gene cassettes and conferred resistance to kanamycin. This is the second class 3 integron reported and the first time that the bla(GES-1) gene cassette has been found on an integron belonging to this class, highlighting the considerable heterogeneity of their genetic environment and the spread of gene cassettes among different classes of integrons.

The possible introduction of rejection criteria for stool cultures (

The Cell-Dyn 3500 instrument could become a sensitive and specific tool in the diagnosis of malaria. The instrument appears to detect malaria-pigment within monocytes and granulocytes. A case of P. vivax malaria in a patient with increased osmotically resistant erythrocytes illustrates the potential of the instrument to detect intraerythrocytic parasites with pigment. However, in most malaria-patients with normal red cell osmotic resistance the observed phenomena seem rather to represent intraleukocytic pigment. This can remain in the circulation of clinically and parasitologically cured individuals and thus may not indicate acute infection. While the instrument can indicate those patients who have been infected a diagnosis of acute malaria must be established independently.

One hundred and thirty-eight isolates of Klebsiella pneumoniae showing resistance to ceftazidime were isolated from different wards of the Hospital de Santa Maria, Lisbon. The genomic DNA of the isolates was analysed by pulsed-field gel electrophoresis (PFGE) and two patterns were predominant. In all isolates the presence of a single large plasmid of about 50 kb suggested that propagation of the outbreak prominently involved plasmid spread. The deduced amino acid sequence indicated the presence of a TEM-10 beta-lactamase. This extended-spectrum beta-lactamase was present among K. pneumoniae isolates, was widely disseminated in different wards and remained persistent as a result of an outbreak involving the dissemination of both the multi-resistance plasmids harbouring the bla gene and the isolates themselves.

The correlation between consumption of antimicrobials in humans and the emergence of resistance in bacteria is complex and has proved difficult to establish. Besides antimicrobial use, many other distinct contributing factors are also involved in the issue. Despite this complexity, there is a substantial body of evidence that the use of antibiotics in prophylaxis and in therapy is associated with the development of resistance in the hospital and in the community. Some examples are reviewed, including increase of resistance in enterobacteriaceae, Pseudomonas aeruginosa, Acinetobacter spp., Streptococcus pneumoniae, Staphylococcus aureus, Coagulase Negative Staphylococci and Streptococcus pyogenes after the use of beta-lactam antibiotics, aminoglycosides, fluoroquinolones and macrolides. Success in reversing the rise of resistant strains has been rarely described. Two examples are highlighted, the reduction in the incidence of nasal carriage of penicillin-resistant pneumococci in Icelandic children, and a significant decline in erythromycin resistance in S. pyogenes after the reduction in the use of macrolides in Finland.

Over a 4-year period, 2020 Staphylococcus aureus strains isolated in Santa Maria Hospital were studied, 26.3% of which were methicillin-resistant (MRSA). The main specimens from which the strains were isolated included pus, blood and sputum/bronchial secretions. Isolation in blood cultures was the most common source among patients from medical units. Antimicrobial susceptibility studies showed that while in methicillin susceptible strains sensitivity to other antimicrobial agents (apart from penicillin resistance) was the rule, in MRSA strains there was resistance to most antibiotics. Only vancomycin was active against all strains. Phage typing showed that 75.5% of the strains were typable with phages at 100 x R.T.D. Among methicillin sensitive strains, a big diversity of phage patterns was observed, including phage groups I, II, III and V, as well as with phage association D11/95. The large majority of MRSA strains were lysed by group III phages, although several distinct patterns were observed. Within these strains, lysis by groups II and V phages was not observed. Plasmid profiling was the least discriminant issue in the characterization of these micro-organisms because most of the strains harboured only one plasmid (or none). These results showed that a dominant MRSA strain did not exist in this hospital, but rather several distinct strains. The importance, as well as the difficulties in controlling the spread of MRSA strains in the present conditions of high prevalence, are highlighted.

In 1994, Microbiology Laboratories of ten Portuguese hospitals analysed isolated microorganisms found in blood and urine samples and studied antimicrobial susceptibilities of the most frequent bacterial pathogens. From 63780 blood samples, the most frequent were Staphylococcus spp. and from 69189 urine samples significant numbers of Escherichia coli, Enterococcus spp., Pseudomonas aeruginosa and Candida spp. were isolated. Escherichia coli strains (c.7000) revealed a low percentage of resistance to antibiotics with the exceptions of ampicillin (48%) and co-trimoxazol (25%). Klebsiella pneumoniae isolates (c.2000) revealed important resistance to ampicillin (98%), cephalotin (31%), co-trimoxazol (38%) and gentamicin (28%), while values for 3rd generation cephalosporins varied among hospitals, with several strains showing phenotype of extended-spectrum beta-lactamase. A great variation in resistance values of P. aeruginosa (c.4000) was found in relation to the antibiotics as well as to the hospitals. Resistance to methicillin in S. aureus (c.6000) was high, reaching an average of 47%, and it was even higher with S. epidermidis (c.3000) and S. haemolyticus (c.650). Only vancomycin was always active against these strains. In E. faecalis (c.2500) resistance was of 2% to ampicillin, 35% to gentamicin, 45% to streptomycin and 1% to vancomycin. E. faecium isolates (c.300) showed the most worrying results with 70% resistance to ampicillin, 42% to gentamicin, 59% to streptomycin and 9% (30 strains isolated in 5 hospitals) to vancomycin. Vancomycin resistant strains were also resistant to all other antibiotics.