Genomic epidemiological analysis of Klebsiella pneumoniae from Portuguese hospitals reveals insights into circulating antimicrobial resistance.

Klebsiella pneumoniae (Kp) bacteria are an increasing threat to public health and represent one of the most concerning pathogens involved in life-threatening infections and antimicrobial resistance (AMR). To understand the epidemiology of AMR of Kp in Portugal, we analysed whole genome sequencing, susceptibility testing and other meta data on 509 isolates collected nationwide from 16 hospitals and environmental settings between years 1980 and 2019. Predominant sequence types (STs) included ST15 (n = 161, 32%), ST147 (n = 36, 7%), ST14 (n = 26, 5%) or ST13 (n = 26, 5%), while 31% of isolates belonged to STs with fewer than 10 isolates. AMR testing revealed widespread resistance to aminoglycosides, fluoroquinolones, cephalosporins and carbapenems. The most common carbapenemase gene was blaKPC-3. Whilst the distribution of AMR linked plasmids appears uncorrelated with ST, their frequency has changed over time. Before year 2010, the dominant plasmid group was associated with the extended spectrum beta-lactamase gene blaCTX-M-15, but this group appears to have been displaced by another carrying the blaKPC-3 gene. Co-carriage of blaCTX-M and blaKPC-3 was uncommon. Our results from the largest genomics study of Kp in Portugal highlight the active transmission of strains with AMR genes and provide a baseline set of variants for future resistance monitoring and epidemiological studies.

ESBL/pAmpC-Producing Escherichia coli Causing Urinary Tract Infections in Non-Related Companion Animals and Humans.

Urinary tract infections (UTI) caused by Escherichia coli are frequently diagnosed in humans and companion animals. Extended-spectrum beta-lactamase (ESBL)- and cephalosporinase (pAmpC)-producing Escherichia coli are worldwide-disseminated and frequently multidrug-resistant, hence leading to treatment failure and public health concerns. This study aimed to characterize and compare ESBL/pAmpC-producing E. coli strains causing community-acquired UTI in companion animals and non-related humans. Third-generation cephalosporin (3GC)-resistant E. coli (companion animals n = 35; humans n = 85) isolated from patients with UTI were tested against 14 antimicrobials following CLSI guidelines. PCR-based assays were used to detect the major E. coli phylogenetic groups, pathogenicity associated-islands (PAIs), virulence genes, and ESBLs/pAmpC resistance genes. ESBL/pAmpC-producing E. coli isolates were typed by multi-locus sequence typing (MLST) and PCR. E. coli strains from companion animals and humans shared two MDR high-risk clonal lineages: ST131 and ST648. To the best of our knowledge, this study reports the first description of E. coli ST131 clade C1-M27 and the clonal lineage ST131 clade A in humans with community-acquired UTI in Portugal. Considering that companion animals with UTI are generally treated at home by the owners, measures should be implemented to avoid the spread of multidrug-resistant high-risk clones to humans and their household environment.

Evidence of Sharing of Klebsiella pneumoniae Strains between Healthy Companion Animals and Cohabiting Humans.

This study aimed to characterize the fecal colonization and sharing of Klebsiella pneumoniae strains between companion animals and humans living in close contact. Fecal samples were collected from 50 healthy participants (24 humans, 18 dogs, and 8 cats) belonging to 18 households. Samples were plated onto MacConkey agar (MCK) plates with and without cefotaxime or meropenem supplementation. Up to five K. pneumoniae colonies per participant were compared by pulsed-field gel electrophoresis (PFGE) after XbaI restriction. K. pneumoniae strains with unique pulse types from each participant were characterized for antimicrobial susceptibility, virulence genes, and multilocus sequence type (MLST). Fecal K. pneumoniae pulse types were compared to those of clinical K. pneumoniae strains from animal and human patients with urinary tract infections (n = 104). K. pneumoniae colonization was detected in nonsupplemented MCK in around 38% of dogs (n = 7) and humans (n = 9). K. pneumoniae strains isolated from dogs belonged to sequence type 17 (ST17), ST188, ST252, ST281, ST423, ST1093, ST1241, ST3398, and ST3399. None of the K. pneumoniae strains were multidrug resistant or hypervirulent. Two households included multiple colonized participants. Notably, two colonized dogs within household 15 (H15) shared a strain each (ST252 and ST1241) with one coliving human. One dog from H16 shared one PFGE-undistinguishable K. pneumoniae ST17 strain with two humans from different households; however, the antimicrobial susceptibility phenotypes of these three strains differed. Two main virulence genotypes were detected, namely fimH-1 mrkD ycfM entB kfu and fimH-1 mrkD ycfM entB kpn These results highlight the potential role of dogs as a reservoir of K. pneumoniae to humans and vice versa. Furthermore, to our best knowledge, this is the first report of healthy humans and dogs sharing K. pneumoniae strains that were undistinguishable by PFGE/MLST.

Klebsiella pneumoniae causing urinary tract infections in companion animals and humans: population structure, antimicrobial resistance and virulence genes.

OBJECTIVES: To characterize the population structure, antimicrobial resistance and virulence genes of Klebsiella spp. isolated from dogs, cats and humans with urinary tract infections (UTIs). METHODS: Klebsiella spp. from companion animals (n = 27) and humans (n = 77) with UTI were tested by the disc diffusion method against 29 antimicrobials. Resistant/intermediate isolates were tested by PCR for 16 resistance genes. Seven virulence genes were screened for by PCR. All Klebsiella pneumoniae from companion animals and third-generation cephalosporin (3GC)-resistant isolates from humans were typed by MLST. All Klebsiella spp. were compared after PFGE XbaI macro-restriction using Dice/UPGMA with 1.5% tolerance. RESULTS: bla CTX-M-15 was detected in >80% of 3GC-resistant strains. K. pneumoniae high-risk clonal lineage ST15 predominated in companion animal isolates (60%, n = 15/25). Most companion animal ST15 K. pneumoniae belonged to two PFGE clusters (C4, C5) that also included human strains. Companion animal and human ST15-CTX-M-15 K. pneumoniae shared a fimH-1/mrkD/entB/ycfM/kfu virulence profile, with a few (n = 4) also harbouring the yersiniabactin siderophore-encoding genes. The hospital-adapted ST11 K. pneumoniae clonal lineage was detected in a cat (n = 1) and a human (n = 1); both were MDR, had 81.1% Dice/UPGMA similarity and shared several virulence and resistance genes. Two 3GC-resistant ST348 strains with 86.7% Dice/UPGMA similarity were isolated from a cat and a human. CONCLUSIONS: Companion animals with UTI become infected with high-risk K. pneumoniae clonal lineages harbouring resistance and virulence genes similar to those detected in strains from humans. The ST15-CTX-M-15 K. pneumoniae clonal lineage was disseminated in companion animals with UTI. Caution must be applied by companion animal caretakers to avoid the spread of K. pneumoniae high-risk clonal lineages.

Clonal relatedness of Proteus mirabilis strains causing urinary tract infections in companion animals and humans.

Proteus mirabilis is a major cause of urinary tract infection (UTI) in humans and companion animals. This study aimed to evaluate the antimicrobial resistance, virulence and clonal relatedness of P. mirabilis isolated from dogs, cats and humans with UTI. P. mirabilis isolated from companion animals (N = 107) and humans (N = 76) with UTI were compared by PFGE analysis after overnight NotI macro-restriction using Dice/UPGMA with a 1.5% tolerance. Strains were characterized for antimicrobial resistance by disk diffusion. Twenty-four resistance genes and four virulence genes were screened by PCR. Thirty-nine clusters (similarity >80%) and 73 single pulse-types were detected. Nine clusters included P. mirabilis isolated from community and hospital patients, including strains with 100% similarity. A high number of clusters (43.6%, n = 17/39) included strains from companion animals and humans. Similarity between some companion animal and human strains varied between 80-100%. One strain from a dog was 100% similar to one human community-acquired P. mirabilis. One P. mirabilis from a cat was found to be 94.7% and 92.4% similar to community and hospital patient strains, respectively. P. mirabilis CMY-2-producers did not cluster all together. Nevertheless, cluster C36 included five P. mirabilis from companion animals (similarity 85.8%-95.7%), of which, four (80%) were multidrug-resistant CMY-2-producers. This study shows that companion animals and humans become infected with closely related P. mirabilis strains. The high number of clusters containing companion animals and human strains points to the zoonotic nature of P. mirabilis. These results underline the potential role of companion animals as reservoirs and in the dissemination of uropathogenic P. mirabilis to humans and vice versa.

Drug-Related Problems Identified in a Sample of Portuguese Institutionalised Elderly Patients and Pharmacists' Interventions to Improve Safety and Effectiveness of Medicines.

BACKGROUND: Currently, people live longer but often with poor quality of life. The decrease in healthy life-years is partly attributable to the institution of polypharmacy to treat various comorbidities. OBJECTIVES: The objectives of the study were to determine the prevalence and nature of drug-related problems (DRPs) in polypharmacy elderly patients residing in nursing homes and to test the acceptability of a pharmacist's intervention. METHODS: An exposure cohort was constituted in three Portuguese nursing homes, where all polypharmacy (five or more medicines) elderly patients (≥65 years of age) were analysed and then a random stratified sample was extracted to be subject to an intervention. Clinical and therapeutic data were collected and analysed for DRPs and classified according to the II Granada Consensus, by a pharmacist-led team. The intervention was the formulation of a pharmacist's recommendations to prescribers addressing clinically relevant DRPs, along with suggestions for therapy changes. RESULTS: The initial sample included 126 elderly patients taking 1332 medicines, where 2109 DRPs were identified. The exposure cohort included 63 patients, with comparable baseline data (p > 0.005). Manifest DRPs occurred in 31.7 % of the intervention group (mainly quantitative ineffectiveness-DRP 4), whereas potential DRPs were identified in 100 % of patients (mainly non-quantitative unsafe-DRP 5). Amongst the DRPs identified, 584 (56.7 %) were reported to prescribers (all types of DRPs) and 113 (11 %) to nurses (only non-quantitative ineffectiveness-DRP 3). A total of 539 pharmacist recommendations were presented to physicians, corresponding to 62 letters sent by mail, each including an average of 8.7 recommendations to solve DRPs present in intervention group (IG) patients. There was a high non-response rate (n = 34 letters; 54.8 %; containing 367 pharmacist recommendations; 68.1 %) and amongst recommendations receiving feedback, only 8.7 % of pharmacist recommendations made were accepted (n = 15). Positive responses were significantly associated with a lower number of recommendations made, whereas a higher number of recommendations increased the odds of no response (p < 0.001). CONCLUSION: A pharmacist-led medication review proved useful in identifying DRPs in elderly polypharmacy nursing home residents. Stronger bonds must be developed between healthcare professionals to increase patient safety in the vulnerable institutionalised elderly population.

Susceptibility patterns of Staphylococcus aureus biofilms in diabetic foot infections.

BACKGROUND: Foot infections are a major cause of morbidity in people with diabetes and the most common cause of diabetes-related hospitalization and lower extremity amputation. Staphylococcus aureus is by far the most frequent species isolated from these infections. In particular, methicillin-resistant S. aureus (MRSA) has emerged as a major clinical and epidemiological problem in hospitals. MRSA strains have the ability to be resistant to most ß-lactam antibiotics, but also to a wide range of other antimicrobials, making infections difficult to manage and very costly to treat. To date, there are two fifth-generation cephalosporins generally efficacious against MRSA, ceftaroline and ceftobripole, sharing a similar spectrum. Biofilm formation is one of the most important virulence traits of S. aureus. Biofilm growth plays an important role during infection by providing defence against several antagonistic mechanisms. In this study, we analysed the antimicrobial susceptibility patterns of biofilm-producing S. aureus strains isolated from diabetic foot infections. The antibiotic minimum inhibitory concentration (MIC) was determined for ten antimicrobial compounds, along with the minimum biofilm inhibitory concentration (MBIC) and minimum biofilm eradication concentration (MBEC), followed by PCR identification of genetic determinants of biofilm production and antimicrobial resistance. RESULTS: Results demonstrate that very high concentrations of the most used antibiotics in treating diabetic foot infections (DFI) are required to inhibit S. aureus biofilms in vitro, which may explain why monotherapy with these agents frequently fails to eradicate biofilm infections. In fact, biofilms were resistant to antibiotics at concentrations 10-1000 times greater than the ones required to kill free-living or planktonic cells. The only antibiotics able to inhibit biofilm eradication on 50 % of isolates were ceftaroline and gentamicin. CONCLUSIONS: The results suggest that the antibiotic susceptibility patterns cannot be applied to biofilm established infections. Selection of antimicrobial therapy is a critical step in DFI and should aim at overcoming biofilm disease in order to optimize the outcomes of this complex pathology.

Potentially inappropriate medications in a sample of Portuguese nursing home residents: Does the choice of screening tools matter?

UNLABELLED: Background Potentially inappropriate medications (PIMs) are often found in high proportion among the elderly population. The STOPP criteria have been suggested to detect more PIMs in European elderly than the Beers criteria. Objective This study aimed to determine the prevalence of PIMs and potential prescribing omissions (PPOs) in a sample of Portuguese nursing homes residents. Setting Four elderly facilities in mainland Portugal Method A descriptive cross-sectional study was used. Elderly polypharmacy patients were included in the study and their medication (registered in patient clinical records) analysed using the Beers (2012 original version and 2008 version adapted to Portugal), STOPP (Screening Tool of Older Person's Prescriptions) and START (Screening Tool to Alert doctors to Right Treatment) criteria. Data were analysed using univariate and bivariate descriptive statistics, considering a confidence interval of 95 %. MAIN OUTCOME MEASURES: Prevalence of PIMs and PPOs. Results The sample included 161 individuals, with a mean age of 84.7 years (SD = 6.35), 68.9 % being female. A total of 807 PIMs and 90 PPOs were identified through the application of the three set of criteria. The prevalence of PIMs using the most recent version of the Beers criteria was 85.1 and 42.1 % for independent and dependent of diagnosis, respectively. The Portuguese adaptation of this same tool indicated a lower prevalence of PIMs, 60.3 and 16.7 %, respectively. The prevalence of PIMs using the STOPP criteria was 75.4 %, whilst the prevalence of PPOs, using START, was 42.9 %. There were significant differences in the mean number of PIMs detected depending on the tool used. (p < 0.001). Conclusions The application of the studied criteria in an elderly sample enabled the identification of a notable amount of PIMs and PPOs, indicating there is room for improving the quality of care. The variation in prevalence indicates careful choice of the tool is a prerequisite for engaging in medication review. Using START/STOPP criteria enabled a more holistic approach to the quality of prescribing in the elderly, highlighting low levels of cardiovascular risk prevention and abuse of psychotropic drugs, aside with system failures largely preventable by electronic prescribing and alert generation.

Molecular typing, virulence traits and antimicrobial resistance of diabetic foot staphylococci.

BACKGROUND: Diabetes mellitus is a major chronic disease that continues to increase significantly. One of the most important and costly complications of diabetes are foot infections that may be colonized by pathogenic and antimicrobial resistant bacteria, harboring several virulence factors, that could impair its successful treatment. Staphylococcus aureus is one of the most prevalent isolate in diabetic foot infections, together with aerobes and anaerobes. METHODS: In this study, conducted in the Lisbon area, staphylococci isolated (n = 53) from diabetic foot ulcers were identified, genotyped and screened for virulence and antimicrobial resistance traits. Genetic relationship amongst isolates was evaluated by pulsed-field-gel-electrophoresis with further multilocus sequence typing of the identified pulsotypes. PCR was applied for detection of 12 virulence genes and e-test technique was performed to determine minimal inhibitory concentration of ten antibiotics. RESULTS: Among the 53 isolates included in this study, 41 Staphylococcus aureus were identified. Staphylococcal isolates were positive for intercellular adhesins icaA and icaD, negative for biofilm associated protein bap and pantone-valentine leucocidin pvl. S. aureus quorum sensing genes agrI and agrII were identified and only one isolate was positive for toxic shock syndrome toxin tst. 36 % of staphylococci tested were multiresistant and higher rates of resistance were obtained for ciprofloxacin and erythromycin. Clonality analysis revealed high genomic diversity and numerous S. aureus sequence types, both community- and hospital-acquired, belonging mostly to clonal complexes CC5 and C22, widely diffused in Portugal nowadays. CONCLUSIONS: This study shows that diabetic foot ulcer staphylococci are genomically diverse, present resistance to medically important antibiotics and harbour virulence determinants. These properties suggest staphylococci can contribute to persistence and severity of these infections, leading to treatment failure and to the possibility of transmitting these features to other microorganisms sharing the same niche. In this context, diabetic patients may become a transmission vehicle for microorganisms' clones between community and clinical environments.

Characterization of multidrug-resistant diabetic foot ulcer enterococci / Caracterización de enterococos multirresistentes aislados de infecciones del pie diabético

INTRODUCTION: Diabetes mellitus is a highly prevalent chronic progressive disease with complications that include diabetic-foot ulcers. METHODS: Enterococci isolated from diabetic-foot infections were identified, evaluated by macro-restriction analysis, and screened for virulence traits and antimicrobial resistance. RESULTS: All isolates were considered multidrug-resistant, cytolysin and gelatinase producers, and the majority also demonstrated the ability to produce biofilms. CONCLUSIONS: These results indicate the importance of enterococci in diabetic-foot infection development and persistence, especially regarding their biofilm-forming ability and resistance to clinically relevant antibiotics

INTRODUCCIÓN: La diabetes mellitus es una enfermedad crónica progresiva de alta prevalencia cuyas complicaciones incluyen úlceras del pie. Procedimiento: Se han identificado enterococos aislados de infecciones del pie diabético, evaluados mediante análisis de macrorrestricción y búsqueda de marcadores de virulencia y de resistencia antimicrobiana. RESULTADOS: Todos los aislados analizados fueron considerados multirresistentes, productores de citolisina y gelatinasa, y la mayoría fueron capaces de formar biofilms. CONCLUSIONES: Estos resultados permiten conjeturar sobre la importancia de los enterococos en el desarrollo y la persistencia de la infección del pie diabético, fundamentalmente debido a la capacidad de formación de biofilm y de resistencia a antibióticos de relevancia clínica

Characterization of multidrug-resistant diabetic foot ulcer enterococci.

INTRODUCTION: Diabetes mellitus is a highly prevalent chronic progressive disease with complications that include diabetic-foot ulcers. METHODS: Enterococci isolated from diabetic-foot infections were identified, evaluated by macro-restriction analysis, and screened for virulence traits and antimicrobial resistance. RESULTS: All isolates were considered multidrug-resistant, cytolysin and gelatinase producers, and the majority also demonstrated the ability to produce biofilms. CONCLUSIONS: These results indicate the importance of enterococci in diabetic-foot infection development and persistence, especially regarding their biofilm-forming ability and resistance to clinically relevant antibiotics.

Polymicrobial biofilms by diabetic foot clinical isolates.

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.

In vitro design of a novel lytic bacteriophage cocktail with therapeutic potential against organisms causing diabetic foot infections.

In patients with diabetes mellitus, foot infections pose a significant risk. These are complex infections commonly caused by Staphylococcus aureus, Pseudomonas aeruginosa and Acinetobacter baumannii, all of which are potentially susceptible to bacteriophages. Here, we characterized five bacteriophages that we had determined previously to have antimicrobial and wound-healing potential in chronic S. aureus, P. aeruginosa and A. baumannii infections. Morphological and genetic features indicated that the bacteriophages were lytic members of the family Myoviridae or Podoviridae and did not harbour any known bacterial virulence genes. Combinations of the bacteriophages had broad host ranges for the different target bacterial species. The activity of the bacteriophages against planktonic cells revealed effective, early killing at 4 h, followed by bacterial regrowth to pre-treatment levels by 24 h. Using metabolic activity as a measure of cell viability within established biofilms, we found significant cell impairment following bacteriophage exposure. Repeated treatment every 4 h caused a further decrease in cell activity. The greatest effects on both planktonic and biofilm cells occurred at a bacteriophage : bacterium input multiplicity of 10. These studies on both planktonic cells and established biofilms allowed us to better evaluate the effects of a high input multiplicity and a multiple-dose treatment protocol, and the findings support further clinical development of bacteriophage therapy.

Evolution of the human immunodeficiency virus type 2 envelope in the first years of infection is associated with the dynamics of the neutralizing antibody response.

BACKGROUND: Differently from HIV-1, HIV-2 disease progression usually takes decades without antiretroviral therapy and the majority of HIV-2 infected individuals survive as elite controllers with normal CD4⁺ T cell counts and low or undetectable plasma viral load. Neutralizing antibodies (Nabs) are thought to play a central role in HIV-2 evolution and pathogenesis. However, the dynamic of the Nab response and resulting HIV-2 escape during acute infection and their impact in HIV-2 evolution and disease progression remain largely unknown. Our objective was to characterize the Nab response and the molecular and phenotypic evolution of HIV-2 in association with Nab escape in the first years of infection in two children infected at birth. RESULTS: CD4⁺ T cells decreased from about 50% to below 30% in both children in the first five years of infection and the infecting R5 viruses were replaced by X4 viruses within the same period. With antiretroviral therapy, viral load in child 1 decreased to undetectable levels and CD4+ T cells recovered to normal levels, which have been sustained at least until the age of 12. In contrast, viral load increased in child 2 and she progressed to AIDS and death at age 9. Beginning in the first year of life, child 1 raised high titers of antibodies that neutralized primary R5 isolates more effectively than X4 isolates, both autologous and heterologous. Child 2 raised a weak X4-specific Nab response that decreased sharply as disease progressed. Rate of evolution, nucleotide and amino acid diversity, and positive selection, were significantly higher in the envelope of child 1 compared to child 2. Rates of R5-to-X4 tropism switch, of V1 and V3 sequence diversification, and of convergence of V3 to a ß-hairpin structure were related with rate of escape from the neutralizing antibodies. CONCLUSION: Our data suggests that the molecular and phenotypic evolution of the human immunodeficiency virus type 2 envelope are related with the dynamics of the neutralizing antibody response providing further support for a model in which Nabs play an important role in HIV-2 pathogenesis.

Wound healing potential of topical bacteriophage therapy on diabetic cutaneous wounds.

Chronic wounds that fail to heal are a common complication of diabetes mellitus and the most common precipitating reason for nontraumatic lower limb amputation. Unfortunately, the bacterial species that cause these infections are becoming more resistant to antibiotics, making them increasingly difficult to treat. We assessed the feasibility of combating chronic bacterial infections with a topically delivered bacteriophage cocktail in two animal models of diabetes mellitus. Microbiological, planimetric, and histological parameters were compared in debrided infected wounds with or without topical bacteriophage treatment. We determined that bacteriophage treatment effectively decreased bacterial colony counts and improved wound healing, as indicated by smaller epithelial and dermal gaps, in Staphylococcus aureus and Pseudomonas aeruginosa infections but was not as effective against Acinetobacter baumannii. Although the improvements were more significant in the rodent model than in the porcine model, our results suggest that topically administered bacteriophage treatment may be effective in resolving chronic infections, especially when applied in conjunction with wound debridement. These findings have important implications for the feasibility of using topical antimicrobial therapies to safely treat chronic infections in diabetes mellitus patients.

Phage endolysins with broad antimicrobial activity against Enterococcus faecalis clinical strains.

Increasing antibiotic resistance of bacterial pathogens has drawn the attention to the potential use of bacteriophage endolysins as alternative antibacterial agents. Here we have identified, characterized, and studied the lytic potential of two endolysins, Lys168 and Lys170, from phages infecting Enterococcus faecalis. Lys168 and Lys170 belong to the cysteine, histidine-dependent amidohydrolases/peptidases (CHAP) and amidase-2 protein families, respectively. Lys168 is quite a unique enterococcal phage endolysin. It shares 95% amino acidic identity with the endolysin of Staphylococcus aureus phage SAP6, which in turn is distantly related to all known CHAP endolysins of S. aureus phages. Lys170 seems to be a natural chimera assembling catalytic and cell-wall-binding domains of different origin. Both endolysins showed a clear preference to act against E. faecalis and they were able to lyse a high proportion of clinical isolates of this species. Specifically, Lys168 and Lys170 lysed more than 70% and 90% of the tested isolates, respectively, which included a panel of diverse and typed strains representative of highly prevalent clonal complexes. Lys170 was active against all tested E. faecalis VRE strains. The quasi specificity toward E. faecalis is discussed considering the nature of the enzymes' functional domains and the structure of the cell wall peptidoglycan.

Novel chimerical endolysins with broad antimicrobial activity against methicillin-resistant Staphylococcus aureus.

Due to their bacterial lytic action, bacteriophage endolysins have recently gained great attention as a potential alternative to antibiotics in the combat of Gram-positive pathogenic bacteria, particularly those displaying multidrug resistance. However, large-scale production and purification of endolysins is frequently impaired due to their low solubility. In addition, a large number of endolysins appear to exhibit reduced lytic efficacy when compared with their action during phage infection. Here, we took advantage of the high solubility of two recently characterized enterococcal endolysins to construct chimeras targeting Staphylococcus aureus. The putative cell wall binding domain of these endolysins was substituted by that of a staphylococcal endolysin that showed poor solubility. Under appropriate conditions the resulting chimeras presented the high solubility of the parental enterococcal endolysins. In addition, they proved to be broadly active against a collection of the most relevant methicillin-resistant S. aureus epidemic clones and against other Gram-positive pathogens. Thus, fusion of endolysin domains of heterologous origin seems to be a suitable approach to design new potent endolysins with changed and/or extended lytic spectrum that are amenable to large-scale production.

Baseline susceptibility of primary HIV-2 to entry inhibitors.

BACKGROUND: The baseline susceptibility of primary HIV-2 to maraviroc (MVC) and other entry inhibitors is currently unknown. METHODS: The susceptibility of 19 HIV-2 isolates obtained from asymptomatic and AIDS patients and seven HIV-1 clinical isolates to the fusion inhibitors enfuvirtide (ENF) and T-1249, and to the coreceptor antagonists AMD3100, TAK-779 and MVC, was measured using a TZM-bl cell-based assay. The 50% inhibitory concentration (IC(50)), 90% inhibitory concentration (IC(90)) and dose-response curve slopes were determined for each drug. RESULTS: ENF and T-1249 were significantly less active on HIV-2 than on HIV-1 (211- and 2-fold, respectively). AMD3100 and TAK-779 inhibited HIV-2 and HIV-1 CXCR4 tropic (X4) and CCR5 tropic (R5) variants with similar IC(50) and IC(90) values. MVC, however, inhibited the replication of R5 HIV-2 variants with significantly higher IC(90) values (42.7 versus 9.7 nM; P<0.0001) and lower slope values (0.7 versus 1.3; P<0.0001) than HIV-1. HIV-2 R5 variants derived from AIDS patients were significantly less sensitive to MVC than variants from asymptomatic patients, this being inversely correlated with the absolute number of CD4(+) T-cells. CONCLUSIONS: T-1249 is a potent inhibitor of HIV-2 replication indicating that new fusion inhibitors might be useful to treat HIV-2 infection. Coreceptor antagonists TAK-779 and AMD3100 are also potent inhibitors of HIV-2 replication. The reduced sensitivity of R5 variants to MVC, especially in severely immunodeficient patients, indicates that the treatment of HIV-2-infected patients with MVC might require higher dosages than those used in HIV-1 patients, and should be adjusted to the disease stage.

Evaluation of the diagnostic performance of the rapid test VIKIA HIV1/2 in a highly complex HIV-1 epidemic.

The rapid test VIKIA HIV1/2 was evaluated in 210 Angolan subjects infected with multiple HIV-1 subtypes and complex recombinant forms and 225 seronegative individuals. All infected subjects tested positive (100% sensitivity); all seronegative subjects tested negative (100% specificity). VIKIA HIV1/2 is highly specific and sensitive even in highly complex epidemics.