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This study aimed to investigate, for the first time, the occurrence and characteristics of extended-spectrum β-lactamase (ESBL)- and carbapenemase-producing Enterobacterales in Cape Verde. A total of 98 inpatients hospitalized at Hospital Universitário Agostinho Neto were screened for rectal colonization. All ESBL- and carbapenemase-producing isolates were tested for antimicrobial susceptibility and characterized by multilocus sequence typing. Mating-out assay followed by PCR-based replicon typing were performed to characterize the plasmids harboring carbapenemase encoding genes. A large proportion of patients carried ESBL- or carbapenemase-producing Enterobacterales (56% and 6%, respectively). Among 93 ESBL-producing isolates, there were mainly Klebsiella pneumoniae (58%) and Escherichia coli (37%). Five different ESBLs were detected, with CTX-M-15 being highly predominant (92%). Six carbapenemase-producing isolates (five E. coli and one K. pneumoniae) were recovered, and all of the OXA-48-like type (four OXA-181, one OXA-48, and one OXA-244). The blaOXA-48 gene was located on an IncFI-type plasmid, the blaOXA-181 gene on IncFI or IncX3 plasmids, and the blaOXA-244 gene was found to be chromosomally located. The five carbapenemase-producing E. coli isolates belonged to five distinct sequence types. This study overall showed a very high prevalence of ESBL-producing Enterobacterales, as well as the emergence of carbapenemase producers in this hospital.

A rapid (3 hours) and reliable multiplex PCR was developed for detecting simultaneously known plasmid-mediated fos genes conferring acquired resistance to fosfomycin. Our technique was tested on a collection of Escherichia coli isolates previously identified as bearing the fosA-, fosC- and fosL-like genes, showing a sensitivity and a specificity of 100%.

Wild birds may be healthy carriers, and therefore, may be involved in the dissemination of clinically relevant antimicrobial-resistant bacteria, such as extended-spectrum β-lactamases (ESBL) and carbapenemase-producing Enterobacteriaceae. This study evaluated whether urban pigeons living in five spots in Lisbon, Portugal, may be colonized and, therefore, constitute potential spreaders of multidrug-resistant bacteria. A total of 100 pigeon fecal samples were collected in different urban areas for the detection of ESBL- or carbapenemase-producing Enterobacteriaceae. All β-lactamase-producing isolates were tested for antimicrobial susceptibility and their genetic backgrounds were characterized by multilocus sequence typing. Of the 100 fecal samples collected, nine ESBL-producing Escherichia coli (9%) were identified. Three isolates carried the blaCTX-M-15 gene, three isolates harbored the blaCTX-M-27 and three isolates carried the blaSHV-12 gene. Genotyping of the nine ESBL-producing E. coli strains revealed seven different sequence types (STs) including ST10, ST131, ST154, ST206, ST1488 (SLV ST10), ST2858 and ST3576, most of which have been already described in humans, animals or in the environment. Urban pigeons constitute a potential source of ESBL genes and may be a transmission vehicle of multidrug-resistant bacteria in the environment.

To evaluate the prevalence of extended-spectrum β-lactamase (ESBL)–producing Enterobacteriaceae fecal carriers at admission in a Portuguese hospital and to determine the epidemiology and antimicrobial resistance patterns of ESBL-producing isolates. During a 2-month period, rectal swabs were collected at hospital admission from 151 at-risk patients. In addition, 48 rectal swabs were obtained from weekly screenings of 37 patients hospitalized for > 48 h. All ESBL/carbapenemase-producing isolates were tested for antimicrobial susceptibility and characterized by PFGE and MLST. The prevalence of ESBL producers at hospital admission was 17% and 24% among at-risk patients hospitalized for > 48 h, while the prevalence of carbapenemase producers was 3% in both cases. Most of the isolates were Escherichia coli (54%) and Klebsiella pneumoniae (41%). The most common ESBL identified was CTX-M-15 (n = 17/34; 50%), followed by CTX-M-27 (n = 10; 29%), CTX-M-33 (n = 4; 12%), SHV-12 (n = 2), and CTX-M-55 (n = 1). The 20 E. coli isolates were distributed into 16 PFGE types and nine sequence types (ST), with 60% of the isolates belonging to ST131. The 15 K. pneumoniae were grouped into 12 PFGE types and nine STs, with three STs (ST17, ST449, ST147) corresponding to 60% of the isolates. A high proportion of isolates showed resistance to ciprofloxacin (86%), trimethoprim-sulfamethoxazole (68%), tobramycin (57%), and gentamicin (43%). All isolates remained susceptible to fosfomycin. A high prevalence of ESBL-producing Enterobacteriaceae was found at hospital admission among at-risk patients and > 50% of the isolates showed resistance to first-line antibiotics for the treatment of lower urinary tract infections, leaving fosfomycin as an alternative.

We aimed to provide updated epidemiologic data on carbapenem-resistant Klebsiella pneumoniae in Portugal by characterizing all isolates (N = 46) recovered during 2013– 2018 in a 123-bed hospital in Lisbon. We identified blaKPC-3 (n = 36), blaOXA-181 (n = 9), and blaGES-5 (n = 8) carbapenemase genes and observed co-occurrence of blaKPC-3 and blaGES-5 in 7 isolates. A single GES-5–producing isolate co-produced the extended-spectrum β-lactamase BEL-1; both corresponding genes were co- located on the same ColE1-like plasmid. The blaOXA-181 gene was always located on an IncX3 plasmid, whereas blaKPC-3 was carried on IncN, IncFII, IncFIB, and IncFIIA plasmid types. The 46 isolates were distributed into 13 pulsotypes and 9 sequence types. All isolates remained susceptible to ceftazidime/avibactam, but some exhibited reduced antimicrobial susceptibility (MIC = 3 mg/L).

Methicillin-resistant Staphylococcus aureus (MRSA) nasal carriage is a major risk factor for infection, namely among populations in the community with inherent prompting factors, such as the homeless. In Portugal, there are no data on S. aureus/MRSA nasal carriage among the homeless community. A total of 84 homeless individuals living in Lisbon (34 with no permanent address and 50 living in shelter) were nasally screened for S. aureus/ MRSA. All isolates were characterized to determine antimicrobial susceptibility and clonal type. A total of 43 (51.2%) S. aureus carriers were identified, including a single individual colonized with MRSA (1.2%). S. aureus carriage rate was higher among individuals with no permanent address (58.8% versus 46%), younger (45.7 ± 12.7 versus 52.5 ± 10.8 years), and with diagnosis of asthma (9% versus 0%). The single MRSA belonged to the EMRSA-15 clone (PFGE D, ST15-SCCmec IVh, and spa type t790). Almost half of the methicillin-susceptible S. aureus (MSSA) isolates (41.9%, n = 18) belonged to two major clones, ST398-t1451 (n = 13) and ST30-t399/t11980/t12808 associated with PFGE I (n = 5). A high proportion of isolates showed non-susceptibility to mupirocin (64%), erythromycin (45%), and fusidic acid (20%) and induced resistance to clindamycin (39%). None of the isolates harboured PVL. Our results suggest that the homeless population of Lisbon does not constitute a reservoir of MRSA in the community, but harbour the highly transmissible ST398-t1451 MSSA lineage.

Objectives: To provide, for the first time, data on the molecular epidemiology of carbapenemase-producing Klebsiella pneumoniae clinical isolates from the northern region of Portugal (Trás-os-Montes and Alto Douro).Methods: A total of 106 carbapenemase-producing K. pneumoniae isolates recovered from clinical samples and rectal swabs between January 2018 and March 2019 were included in this study. All isolates were characterized by antimicrobial susceptibility, identification of resistance determinants, pulsed-field gel electrophoresis (PFGE), multilocus sequence typing (MLST), and plasmid analysis.Results: The most common carbapenemase identified was KPC-2 (91%), followed by OXA-48 (9%). The blaKPC- 2 gene was carried onto IncN (60%) and IncF (40%) plasmid types, whereas the blaOXA-48 gene was mainly located on the IncL (90%) incompatibility group. Molecular characterization distributed the 106 isolates into 29 PFGE types and 21 sequence types (STs), but three clones included 50% of the isolates: PFGE A-ST147- KPC-2 (29%), B-ST15-KPC-2 (15%), and C-ST11-OXA-48 (6%). Antimicrobial resistance rates were the following: ciprofloxacin (76%), trimethoprim– sulfamethoxazole (75%), tobramycin (62%), gentamicin (34%), amikacin (25%), tigecycline (21%), fosfomycin (10%), and colistin (7%). None of the colistin-resistant isolates harboured mcr genes. All isolates remained susceptible to ceftazidime/avibactam, but 10% presented elevated MICs (3 and 4 mg/L).Conclusions: KPC-2 was the predominant carbapenemase among K. pneumoniae isolates currently circulating at this hospital from northern Portugal, followed by OXA-48. These data contrast with those obtained from the rest of the country, where KPC-3 predominates. This study showed a polyclonal structure of KPC-2-producing K. pneumoniae isolates with a predominance of the ST147 and ST15 clones.

Objectives: Methicillin-resistantStaphylococcus aureus (MRSA) remains a major human pathogen. MRSA decolonisation strategies frequently combine chlorhexidine baths and mupirocin nasal ointment. Although MRSA remains widespread in Portuguese hospitals, information regarding resistance to biocides and mupirocin is scarce. We evaluated the prevalence of biocide resistance genes and chlorhexidine and mupirocin non-susceptibility in a representative and well-characterised collection of MRSA isolated in Portuguese hospitals during a 31-year period (1985–2016). Methods: Prevalence of five biocide resistance genes (lmrS, mepA, sepA, qacAB and smr) was determined by PCR. Antibiotic susceptibility was assessed by disk diffusion and by MIC determination using broth microdilution (chlorhexidine) and Etest (mupirocin). Results: Chromosomal genessepA and mepA were detected in all isolates, while lmrS was found in 87.1%. The prevalence of plasmid-borne genes was significant for qacAB (22.4%), associated with the Iberian (ST247-I/IA) clone (P < 0.0001), and low for smr (1.0%) detected among isolates belonging to the ST239-III/IIIvariant clone. Chlorhexidine non-susceptibility (MIC ≥ 4 mg/L) was observed in two isolates belonging to the EMRSA-15 clone (ST22-IV). Non-susceptibility to mupirocin (MIC > 1 mg/L) was significant (15.4%; n = 31) and mainly found among isolates of the EMRSA-15 clone (P < 0.0001; n = 29). One isolate presented low-level mupirocin resistance (MIC = 32 mg/L), and two missense mutations N213D (A637G) and V588F (G1762T) were identified in the ileS gene. Conclusion: Concerningly, we detected a high prevalence of biocide resistance genes and an association of mupirocin and chlorhexidine non-susceptibility with the dominant EMRSA-15 clone in Portuguese hospitals.

Aims To undertake a prospective analysis of the occurrence of colistin-resistant and extended-spectrum s-lactamase (ESBL)-producing Enterobacterales colonizing pigs at two farms in Portugal, and to evaluate the putative correlations with usage of different antibiotics. Materials and methods One hundred and two faecal samples recovered from two different Portuguese pig farms were screened for polymyxin-resistant and ESBL-positive Enterobacterales. The authors had undertaken a study at one of the farms previously, but the use of colistin has since been banned; zinc oxide and amoxicillin are used as prophylactic and curative drugs, respectively, at this farm. The other farm included in this study used zinc oxide alone. Results Ninety-three ESBL-producing isolates (62 Escherichia coli, 29 Klebsiella pneumoniae, one Enterobacter aerogenes and one Enterobacter cloacae) and 17 colistin-resistant isolates (12 E. coli, four K. pneumoniae and one E. cloacae) were recovered. Among the ESBL producers, the majority (84%) produced CTX-M-15, while the others produced CTX-M-1 or CTX-M-9. Many different strain and plasmid backgrounds were identified, ruling out a massive dissemination of one major clone. In total, 17 colistin-resistant isolates were recovered, all from the first farm. All produced MCR-1, corresponding to 12 E. coli (10 clones) and three K. pneumoniae (two clones). The MCR-1 producers were all recovered from the farm where colistin had been used 2 years previously. Conclusion This study showed a surprisingly high rate of CTX-M-15 producers at two Portuguese pig farms. A link was found between antibiotic selective pressure (s-lactam or polymyxin) and the corresponding resistance rate.

Summary Background The prevalence of nosocomial meticillin-resistant Staphylococcus aureus (MRSA) was previously estimated as 23% in a paediatric hospital in Luanda, Angola and 18% in a general hospital in Sao Tome and Principe. Aim To evaluate the prevalence of S. aureus/MRSA colonization among hospitalized children and their parents at two hospitals in Angola and Sao Tome and Principe. Methods In 2017, 127 hospitalized children and 129 of their parents had nasal swabs for S. aureus/MRSA carriage in the two countries. The isolates were tested for the presence of the mecA and Panton-Valentine leukocidin (PVL) genes, and characterized by pulsed-field gel electrophoresis (PFGE), spa typing, multi-locus sequence typing and SCCmec typing. Findings Twenty of 127 children (15.7%) and 13 of 129 parents (10.1%) were MRSA nasal carriers. Three lineages comprised 88% of the MRSA isolates: (i) PFGE A-ST5-SCCmec IVa (N=15; 45%), associated with spa type t105, recovered in Angola alone; (ii) PFGE N-ST8-IV/V (N=7; 21%), associated with spa types t008/t121, recovered in Sao Tome and Principe alone; and (iii) PFGE B-ST88-IVa (N=7; 21%), associated with spa types t325/t786, present in both countries. Fifteen child/guardian pairs were colonized with identical MRSA (N=8) or meticillin-susceptible S. aureus (N=7) strains. PVL was detected in 25% of isolates, including two MRSA (ST30-V and ST8-IVa). Conclusion Hospitalized children and their parents are important reservoirs of MRSA. Infection control measures should focus on parents in order to minimize the spread of MRSA to the community.

Multidrug-resistant bacteria pose a major threat to effective antibiotics and alternatives to fight multidrug-resistant pathogens are needed. We synthetized molybdenum oxide (MoO3) nanoparticles (NP) and determined their antibacterial activity against 39 isolates: (i) eight Staphylococcus aureus, including representatives of methicillin-resistant S. aureus epidemic clones; (ii) six enterococci, including vancomycin-resistant isolates; and (iii) 25 Gram-negative isolates (Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa, Acinetobacter baumannii, Enterobacter cloacae), including extended spectrum beta-lactamases and carbapenemases producers. All isolates showed a MoO3 NP MIC of 700–800 mg l−1. MoO3 NP produced a clear inhibition zone for S. aureus and all Gram-negative isolates at concentrations ≥25 mg ml−1 and ≥50 mg ml−1 for enterococci. When the NP solutions were adjusted to pH ~7, the biocidal activity was completely abolished. MoO3 NP create an acidic pH and show a universal antimicrobial activity against susceptible and resistant isolates belonging to the most relevant bacterial species responsible for hospital-acquired infections.

Methicillin-resistant Staphylococcus aureus (MRSA) can survive for long periods on inanimate objects, and therefore, environmental surfaces constitute an important reservoir for dissemination. However, there is no standardized method for the detection of MRSA from environmental surfaces. The aim of the present study was to evaluate different screening methods to detect environmental MRSA contamination. A total of 294 samples were obtained from inanimate surfaces at a hospital in Luanda, Angola, and a hospital in São Tomé and Príncipe, by three different methodologies: (1) sterile swabs moistened in saline solution, (2) sterile cotton gauzes moistened in tryptic soy broth, and (3) commercial premoistened sterile sponges (polywipes). After a broth enrichment step, all samples were plated onto tryptic soy agar and chromogenic selective media for S. aureus and for MRSA. The S. aureus isolates were characterized by pulsed-field gel electrophoresis, spa typing, multilocus sequence typing, and SCCmec typing. Comparing the three screening methods, gauzes were the most effective (16 S. aureus out of 98 samples; 16.3%), followed by polywipes (4/98; 4.1%) and swabs (3/98; 3.1%). Moistened gauzes were the most sensitive method (p 0.00001), while screening with swabs was the least efficient (p = 0.00002). The majority of the MRSA isolates (75%) belonged to the main clonal types previously found among patients and healthcare workers in the same hospitals: ST5-IVa (n = 7; 44%) and ST88-IVa (n = 5; 31%). The finding of MRSA on environmental surfaces is dependent on the screening methodology. Moistened gauzes followed by a broth enrichment step proved to be the most sensitive methodology compared to polywipes and swabs.

High rates of trimethoprim/sulfamethoxazole (SXT) resistance, a combination of two antifolate antibiotics trimethoprim (TMP) and sulfamethoxazole (SMZ), have been reported among Staphylococcus aureus isolates in Portuguese-speaking African countries. Our study aimed to evaluate the occurrence of TMP resistance markers in major SXTR methicillin-resistant S. aureus (MRSA) clones from these countries. We accessed also different fitness traits that could explain the success of these isolates over the Brazilian MRSA (the most successful SXTR MRSA clone worldwide but never identified in these countries). Minimum inhibitory concentrations for SXT, TMP and SMZ were determined, and genes encoding TMP resistance (dfrG, dfrA, dfrK and dfrB) were searched. Representatives of the Brazilian clone and of the major MRSA African clones were evaluated for their fitness by individual growth curves, competition assays, survival under desiccation, autolytic activity, resistance to oxidative stress, and also growth at high osmolarity and in acid and alkaline environments. Although all African isolates showed high-level resistance to TMP, the majority presented hetero-resistance to SXT. TMP resistance was linked to the presence of dfrG (78%), dfrA (19%) or both (3%) genes. Compared to the Brazilian clone, the African isolates showed higher growth rates and autolytic activity, and better survival to desiccation and alkaline conditions. Since isolates exhibiting SXT hetero-resistance are frequent in Africa, the implementation of standardized guidelines to detect this phenomenon is of major interest. The predominant MRSA clones in Portuguese-speaking African countries likely possess significant advantages over other clones, such as the Brazilian MRSA, that may explain their epidemiological success.

Objectives Data on baseline drug resistance is important in informing future antimicrobial stewardship programs. So far, no data on the antimicrobial drug resistance of clinical isolates was available for the African archipelago of Cabo Verde. Methods We have performed a retrospective analysis over five-years (2013-17) of the antimicrombial drug susceptibility profiles of clinical isolates in the two main hospitals of Cabo Verde. For Escherichia coli and Staphylococcus aureus, representing respectively 47% and 26% of all clinical isolates, the antimicrobial drug resistance profile was reported for six representative drugs. Results For E. coli we detected an increase in resistance to ampicillin, amoxicillin/clavulanic acid, ceftriaxone, ciprofloxacin, and trimethoprim-sulfamethoxazole and for S. aureus to methicillin, erythromycin and trimethoprim-sulfamethoxazole. This increase in both the most commonly isolated bacterial pathogens is of alarm as it might compromise empirical treatment in a setting with limited access to laboratory testing. Conclusions When compared to the published low resistance rates in carriage isolates, the more alarming situation in clinical isolates for S. aureus might encourage antimicrobial stewardship programs to reduce MRSA in the hospital settings, possibly as part of the Cabo Verdean national plan against antimicrobial drug resistance.

Objectives To decipher the genetics of acquisition of carbapenemase-encoding genes identified in two carbapenem-resistant Enterobacteriaceae recovered from a single patient in Portugal. Methods Carbapenemase genes were searched by PCR assays and mating-out assays were performed to further characterize the plasmid support of the carbapenemase genes. Genetic characterization of the plasmid supports was performed by whole-plasmid sequencing using the Illumina technology. Results We identified here two NDM-1-producing isolates, namely a Morganella morganii and a Proteus mirabilis, sharing the same blaNDM-1-positive plasmid. This 154 kb plasmid belonged to the IncA/C2 type, recently renamed IncC, and co-harboured two AmpC β-lactamase genes, namely blaCMY-4 and blaDHA-1, in addition to the 16S rRNA methylase gene armA encoding high-level resistance to aminoglycosides. In addition, the M. morganii isolate produced the CTX-M-33 extended-spectrum β-lactamase possessing weak carbapenemase activity, encoded by another plasmid. Conclusions We showed here that, in addition to KPC-type and OXA-181 carbapenemases, which have been identified as widespread in this country, another concern is the emergence of NDM-1-producing enterobacterial isolates in Portugal. We demonstrated here the in vivo plasmid transfer of a blaNDM-1-positive plasmid leading to dissemination of this carbapenemase gene within different enterobacterial species in a single patient.

BackgroundIn 2016, very high rates of methicillin-resistant Staphylococcus aureus (MRSA)-ST398 (99%) were found in Portuguese pig farms that used colistin, amoxicillin, and zinc oxide as feed additives. Since then, farms A and B banned the use of colistin, and farm C banned the use of both antibiotics.ObjectiveThe aim of the present study was to evaluate the impact of the ban of colistin and amoxicillin on pig MRSA carriage rates, clonal types and antimicrobial resistance, compared to the results obtained in 2016.MethodsIn 2018, 103 pigs (52 from farm B using amoxicillin only as a feed additive and 51 from farm C where no antibiotics were included in the feed regimen) were nasally swabbed for MRSA colonization. Isolates were tested for antimicrobial susceptibility, and characterised by spa typing, SCCmec typing and MLST. Whole genome sequencing (WGS) was performed for representative isolates.ResultsOverall, 96% of the pigs swabbed in 2018 carried MRSA, mostly ST398-SCCmec V-spa types t011/t108. MRSA from pigs not receiving antibiotics in the feed regimen showed susceptibility to a higher number of antibiotics, namely erythromycin, ciprofloxacin, gentamicin, and chloramphenicol. Notably, most of these isolates (n = 52) presented an unusual erythromycin-susceptibility/clindamycin-resistance phenotype. WGS showed that these isolates lacked the erm and the lnu genes encoding resistance to macrolides and lincosamides, respectively, but carried the vgaALC gene encoding resistance to lincosamides, which is here firstly identified in S. aureus ST398.ConclusionAfter two years the ban of colistin and amoxicillin as feed additives had no significant impact on the MRSA nasal carriage rates. Nevertheless, the MRSA strains circulating in those farms showed resistance to a lower number of antibiotic classes.

This study aimed to evaluate the prevalence of carbapenemase-producing Enterobacteriaceae in Luanda, Angola. A total of 157 rectal samples were collected from children visiting a pediatric hospital in Luanda in March 2015. Fifty-seven imipenem-nonsusceptible enterobacterial isolates were recovered, most of which were non-clonally related. The bla OXA-181 (50/57) and bla NDM-1 (7/57) carbapenemase genes were identified. Notably, OXA-181-producing Escherichia coli isolates rarely coproduced extended-spectrum β-lactamases and consequently remained susceptible to broad-spectrum cephalosporins. The bla OXA-181 gene was always located on an IncX3 plasmid, while the bla NDM-1 gene was located on either IncFIA or IncA/C plasmids. The study identified a high prevalence of OXA-181 among hospitalized children in Angola.

OBJECTIVES Recent surveillance of MRSA colonizing patients and healthcare workers in two African countries (Angola and Sao Tome and Principe) reported the frequent recovery of oxacillin-susceptible MRSA (OS-MRSA): Staphylococcus aureus strains that gave positive results with the mecA DNA probe, but had low oxacillin MIC values characteristic of susceptible S. aureus. This apparent dissociation of the drug-resistant phenotype from mecA-the primary genetic determinant of resistance-prompted us to perform a more detailed analysis on nine of the African OS-MRSA strains. METHODS Oxacillin MIC values were determined by Etest and population analysis profiles with and without induction of the stringent stress response by mupirocin. Biochemical profiling using SDS-PAGE followed by western blotting was used for the detection of PBP2A protein produced. RESULTS Cultures of the African MRSA strains (ST88-IVa and ST8-V) showed heterogeneous oxacillin resistance in which the majority of cells exhibited low oxacillin MICs (≤0.75 mg/L), but highly resistant subpopulations were also present with oxacillin MIC values up to several hundred mg/L and with frequencies of 10(-4) to 10(-6). The same strains after induction of the stringent stress response by mupirocin 'converted' the heterogeneous phenotypes into a more homogeneous and higher level resistance. After induction by oxacillin and mupirocin, each of the nine African OS-MRSA strains produced PBP2A-the protein product of mecA. CONCLUSIONS The resistant phenotype of OS-MRSA resembles the phenotypes of historically early MRSA clones. The nature of genetic determinants responsible for the heterogeneous phenotypes of OS-MRSA remains to be determined.

We assessed the prevalence of six biocide resistance genes among 82 methicillin-resistant Staphylococcus aureus (MRSA) and 219 methicillin-susceptible S. aureus (MSSA) isolates from three African countries; the prevalence was very high for sepA (95.3%), mepA (89.4%), and norA (86.4%), intermediate for lmrS (60.8%) and qacAB (40.5%), and low for smr (3.7%). A significant association between biocide resistance genes and antibiotic resistance was observed, and a new cutoff MIC of ≥1 mg/liter for chlorhexidine nonsusceptibility was defined.

The occurrence of resistance to last-resort antibiotics was evaluated among Enterobacteriaceae isolates recovered from hospitalized children in a remote African archipelago, São Tomé and Príncipe, where there is limited access to those antibiotics. Fifty patients were screened for colonization by carbapenem-, pan- aminoglycoside-, or polymyxin-resistant Enterobacteriaceae. A total of 36 isolates (including 30 Escherichia coli and 4 Klebsiella pneumoniae) were recovered from 23 patients, including 26 isolates harboring the blaOXA-181 carbapenemase gene, a single isolate harboring the 16S rRNA methylase gene rmtB encoding pan-resistance to aminoglycosides, and 8 isolates coharboring both genes. A single isolate possessed the plasmid-borne colistin resistance gene mcr-1. A high clonal relationship was found for OXA-181-producing E. coli (4 clones), and conversely, three of the four OXA-181-producing K. pneumoniae isolates were clonally unrelated. This study overall showed a high prevalence of resistance to last-resort antibiotics in this country, where no epidemiological data were previously available.

Acquired 16S rRNA methylases (RMTases) conferring pan-drug resistance to aminoglycosides were searched among enterobacterial isolates recovered in Angola. A total of 36 hospitalized children were screened for rectal colonization using the Superaminoglycoside selective medium. Twenty-two pan-aminoglycoside-resistant enterobacterial isolates were recovered, all of which produced RMTases, i.e., RmtB, ArmA, and RmtC. Highly diverse genetic backgrounds were identified for Escherichia coli and Klebsiella pneumoniae isolates, most of which coproduced carbapenemases NDM-1 or NDM-5, respectively.

The mcr-1 (mobile colistin resistance 1) gene, which encodes phosphoethanolamine transferase, has been recently identified as a source of acquired resistance to polymyxins in Escherichia coli. Using the SuperPolymyxin selective medium, we prospectively screened 100 pigs at 2 farms in Portugal for polymyxin-resistant Enterobacteriaceae and recovered 98 plasmid-mediated MCR-1–producing isolates. Most isolates corresponded to nonclonally related E. coli belonging to many sequence types; we also found 2 Klebsiella pneumoniae sequence types. The mcr-1 gene was carried on IncHI2 or IncP plasmid backbones. Our finding of a high rate of MCR-1 producers on 2 pig farms in Portugal highlights the diffusion of that colistin-resistance determinant at the farm level. The fact that the pigs received colistin as metaphylaxis in their feed during the 6 weeks before sampling suggests selective pressure.

Background Staphylococcus aureus is a major human pathogen that can colonize healthy people mainly in the anterior nares. The aim of the present study was to evaluate S. aureus nasal colonization over time among Portuguese nursing students, including methicillin-resistant S. aureus (MRSA). Methods and findings In this longitudinal cohort study, we collected 280 nasal swabs from nursing students at 14 time points over four years of schooling (2012-2016). The isolates were characterized by pulsed-field gel electrophoresis (PFGE), spa typing, multilocus sequence typing (MLST), and SCCmec typing for MRSA. Among 47 students, 20 (43%) carried methicillin-susceptible S. aureus (MSSA) at admission, but none was colonized with MRSA. A total of 19 students (40%) became colonized after exposure during the nursing training, out of which five carried MRSA. Overall, 39 students (83%) had S. aureus detected at least once during the study period. Among the 97 MSSA isolates, most (65%) belonged to four clones: PFGE A-ST30 (21%), B-ST72 (20%), C-ST508 (13%), and D-ST398 (11%). Three of the five MRSA carriers were colonized with the predominant clone circulating in Portuguese hospitals (ST22-IVh) and two with ST3162-II. Colonization of nursing students was highly dynamic with continuous appearance of strains with distinct PFGE types in the same individual. Conclusions A considerable proportion of students became colonized by S. aureus, including MRSA, during the nursing education, evidencing this population represents an important reservoir of S. aureus. Therefore, education on infection control measures in nursing schools is of major importance.

Multidrug-resistant (MR) Gram-negative (GN) pathogens pose a major and growing threat for healthcare systems, as therapy of infections is often limited due to the lack of available systemic antibiotics. Well-tolerated antiseptics, such as octenidine dihydrochloride (OCT), may be a very useful tool in infection control to reduce the dissemination of MRGN. This study aimed to investigate the bactericidal activity of OCT against international epidemic clones of MRGN. A set of five different species (Escherichia coli, Klebsiella pneumoniae, Enterobacter cloacae, Acinetobacter baumannii, and Pseudomonas aeruginosa) was studied to prove OCT efficacy without organic load, under “clean conditions” (0.3 g/L albumin) and under “dirty conditions” (3 g/L albumin + 3 mL/L defibrinated sheep blood), according to an official test norm (EN13727). We used five clonally unrelated isolates per species, including a susceptible wild-type strain, and four MRGN isolates, corresponding to either the 3MRGN or 4MRGN definition of multidrug resistance. A contact time of 1 min was fully effective for all isolates by using different OCT concentrations (0.01% and 0.05%), with a bacterial reduction factor of >5 log10 systematically observed. Growth kinetics were determined with two different wild-type strains (A. baumannii and K. pneumoniae), proving a time-dependent efficacy of OCT. These results highlight that OCT may be extremely useful to eradicate emerging highly resistant Gram-negative pathogens associated with nosocomial infections.

The nosocomial prevalence of methicillin-resistant Staphylococcus aureus (MRSA) in Portugal is close to 50% and remains one of the highest in Europe. MRSA reservoirs in the animal setting in Portugal have been very poorly investigated, namely among animal husbandry. A total of 52 samples (nasal, inguinal region, and milk) were obtained from bovine animals and analyzed for the presence of S. aureus. The isolates were characterized by pulsed-field gel electrophoresis (PFGE), spa typing, SCCmec typing, and multilocus sequence typing and tested for antimicrobial susceptibility, presence of mecA and mecC genes, and virulence determinants. Overall, 54% of the screened animals were colonized with S. aureus in at least one body site. Notably, S. aureus nasal carriage followed an increasing trend with animal age (p = 0.0006). None of the isolates harbored the mecA or mecC genes. Resistance to penicillin, rifampicin, and tetracycline was observed in 24%, 18%, and 6% of the isolates, respectively. The isolates were distributed into three clonal lineages: PFGE type A, spa type t1166, ST1247-CC133 (43%), PFGE B-t267-ST352-CC97 (30%), and PFGE C-t091-ST7-CC7 (27%). CC133 was associated to older animals (p = 0.0025), whereas CC97 was isolated from calves (p = 0.0016). Virulence determinants commonly found in mastitis were widely detected in carriage isolates: lukDE and hlgv (100%), hlb (76%), and lukM (35%). Although healthy bovines do not represent a MRSA reservoir in Portugal, they are mainly colonized with S. aureus pathogenic lineages associated to mastitis in cattle (CC97 and CC133).

Background Although livestock-associated ST398 methicillin-resistant Staphylococcus aureus (MRSA) has been widely reported in different geographic regions, MRSA carriage studies among healthy pigs in Portugal are very limited. Methods and findings In total, 101 swine nasal samples from two Portuguese farms were screened for MRSA. In addition five swine workers (including one veterinary and one engineer) and four household members were nasally screened. The isolates were characterized by spa typing, SCCmec typing and MLST. All isolates were tested for antimicrobial susceptibility, presence of mecA and mecC genes, and virulence determinants. MRSA prevalence in swine was 99% (100/101), 80% (4/5) in swine workers and 25% (1/4) in household members. All isolates belonged to ST398 distributed over two spa types–t011 (57%) and t108 (42%). SCCmec type V was present in most of the isolates (n = 95; 82%) while 21 isolates amplified the mecA gene only and were classified as nontypeable. The majority of the isolates were resistant to tetracycline (100%), clindamycin (97%), erythromycin (96%), chloramphenicol (84%) and gentamycin (69%). Notably, 12% showed resistance to quinupristin-dalfopristin (MICs 3–8 μg/mL). Beta-hemolysin (81%) and gamma-hemolysin (74%) were the unique virulence determinants detected. None of the isolates harboured PVL or mecC gene. Conclusions This study showed a massive occurrence of ST398-MRSA in two independent swine farms, highlighting its establishment among healthy pigs in Portugal.

The Check Your Biosignals Here initiative (CYBHi) was developed as a way of creating a dataset and consistently repeatable acquisition framework, to further extend research in electrocardiographic (ECG) biometrics. In particular, our work targets the novel trend towards off-the-person data acquisition, which opens a broad new set of challenges and opportunities both for research and industry. While datasets with ECG signals collected using medical grade equipment at the chest can be easily found, for off-the-person ECG data the solution is generally for each team to collect their own corpus at considerable expense of resources. In this paper we describe the context, experimental considerations, methods, and preliminary findings of two public datasets created by our team, one for short-term and another for long-term assessment, with ECG data collected at the hand palms and fingers.

We previously synthetized molybdenum oxide (MoO3) nanoparticles (NP) and showed their antibacterial activity against a representative collection of the most relevant bacterial species responsible for hospital-acquired infections, including Staphylococcus aureus. The aim of the present study was to prepare and characterize a novel coating with these MoO3 NP, confirm its mechanical stability, and investigate its biocidal effect to reduce S. aureus contamination on inanimate surfaces. In addition, the novel MoO3 NP coating was compared to a silver (Ag) NP coating synthetized by the same procedure. The MoO3 and Ag NP coatings were characterized in terms of their chemical structure by FT-IR, surface morphology by scanning electron microscopy, and mechanical properties by tensile and adhesion tests. The antimicrobial activity of the coatings was tested by following the loss of viability of S. aureus after 6h, 24h, 48h, and 72h exposure. MoO3 and Ag coatings exhibited surfaces of comparable morphologies and both presented elastomeric properties (tensile strength of ~420 kPa, Young's modulus of ~48 kPa, and maximum elongation of ~12%), and excellent (classification of 5B) adhesion to glass, steel and polystyrene surfaces. The two coatings exhibited a good antibacterial activity (R) against S. aureus over time (RMoO3 = 0.2-0.81; RAg = 0.61-2.37), although the effect of the Ag NP coating was more pronounced, especially at 72h (RMoO3 = 0.81 vs RAg = 2.37). Noteworthy, contrary to the Ag NP coating, the MoO3 NP coating was colourless and transparent, avoiding undesired unaesthetic effects. The synthetized coating with NP of MoO3, which has low toxicity to humans, capability of biodegradation, and rapid excretion, can be applied onto most standard materials and therefore is a promising tool to reduce S. aureus contamination on usual inanimate surfaces found in healthcare and community environments.

Currently, methicillin-resistant Staphylococcus aureus (MRSA) is a universal threat. After being well established in the healthcare setting, it has emerged in the community among people with no risk factors for MRSA acquisition, therefore imposing a new threat. The subsequent detection of MRSA colonizing or infecting animals as well as in food of animal origin was of major concern, revealing new reservoirs for MRSA. The major MRSA clonal lineages circulating in the different settings, i.e. in hospitals, in the community and among animals, are described here, differentiating between clones colonizing companion and food-chain animals. Particular attention is given to the widely spread livestock-associated MRSA clonal complex (CC) 398, which is mainly associated with professional exposure but may be of high pathogenicity. The recent detection of a mecA homologue, designated mecC, with a wide geographical distribution in Europe, and including a large diversity of hosts (food-chain, companion and wildlife animals and also detected in water samples) adds to the threat. Domestication as well as globalization of the livestock industry have intensified exchanges between human and animal bacteria. We report here several cases of transmission of MRSA between companion or food-chain animals and humans, as well as some MRSA clones of human origin that have adapted to new animal hosts eventually by losing useless virulence factors or acquiring new mobile genetic elements.

Although Staphylococcus aureus is a major cause of outbreaks in neonatal intensive care units (NICUs), there are no studies on the epidemiology of S. aureus isolates responsible for infection in Portuguese NICUs. Between July 2005 and December 2007, a total of 54 methicillin susceptible S. aureus (MSSA) isolates were recovered from 16 infected infants, parents, health care workers (HCWs), and the environment in a level III NICU. Isolates were characterized by pulsed-field gel electrophoresis (PFGE), spa typing, and multilocus sequence typing. Virulence determinants were detected by multiplex polymerase chain reaction. Three major MSSA clones were endemic in the NICU, representing 70% (n=38) of the isolates: PFGE type A-ST5 (n=17); type B-ST30 (n=12); and type C-ST1 (n=9). Leukotoxins and hemolysins were present in all isolates, although none of them carried PVL. HCWs, plastic folders protecting clinical files, and mothers' nipples were identified as potential reservoirs and/or vehicles of dissemination of S. aureus. Consequently, additional infection control measures were implemented in this NICU.

In order to evaluate the incidence of community-associated methicillin-resistant Staphylococcus aureus (CA-MRSA) in Portugal, we analyzed a collection of 38 S. aureus isolates recovered from 30 children attending the pediatric emergency department of a central hospital in Lisbon due to skin and soft tissue infections. Molecular characterization identified seven clonal lineages among the 35 methicillin-susceptible S. aureus (MSSA) isolates, of which the major lineage PFGE A/t159/ST121 included 63% of the isolates. The three MRSA isolates belonged to the Pediatric clone PFGE D/t535/ST5-IV (n = 2) and to the European CA-MRSA clone PFGE G/t044/ST80-IVc (n = 1). All isolates harbored several virulence factors, namely, leukocidins. Panton–Valentine leukocidin (PVL) was produced by isolates from five MSSA lineages and by the ST80 MRSA. Of interest, this is the first reported isolation of CA-MRSA ST80 in Portugal.

In order to obtain insights into the methicillin-resistant Staphylococcus aureus (MRSA) population structure in the Azores archipelago, 106 MRSA isolates were collected from patients attending an Azorean central hospital between January 2007 and February 2008. Antimicrobial resistance was determined for all isolates. Molecular typing was performed by pulsed-field gel electrophoresis (PFGE), spa typing, multilocus sequence typing (MLST), staphylococcal chromosome cassette mec (SCCmec) typing and the presence of Panton-Valentine leukocidin (PVL). The majority of the isolates (87%, n = 92) belonged to the EMRSA-15 clone (ST22, SCCmec-IVh), followed by the Pediatric clone (ST5-VI/IVc) (11%, n = 12). The Berlin clone (ST45-IVa) and a new clone (spa type t1839, ST1339 and SCCmec V variant) were represented by single isolates. All of the isolates carried SCCmec types IV, V or VI and a non-multiresistant antibiotic profile, resembling the currently emerging community MRSA. Moreover, PVL was described for the first time to be associated with the Pediatric clone carrying SCCmec type VI. We provided the first description of the population structure of MRSA in the Azores islands, which seems to be shaped by genetic events occurring locally, as well as by the regular population exchange between the islands, continental Portugal, the United Kingdom and the United States.

A total of 629 nonduplicate methicillin-resistant Staphylococcus aureus MRSA isolates were recovered between June and November 2006 from 11 hospitals located in different areas of Portugal. Selected isolates ( n = 271, 43%) were typed by pulsed-field gel electrophoresis (PFGE), representatives of which were additionally characterized by spa typing, multilocus sequence typing, staphylococcal cassette chromosome mec (SCC mec ) typing, and the presence of Panton-Valentine leukocidin (PVL). The 271 isolates were classified into 13 different clonal types. Three pandemic clones included the majority ( n = 241, 88%) of the isolates and were observed in several hospitals: (i) EMRSA-15 (54%)—PFGE type A, ST22, spa type t022, SCC mec IV—was found in the 11 hospitals studied and was identified as the major clone in seven of them; (ii) the New York/Japan clone (17%)—PFGE B, ST5, spa type t067, SCC mec II—was identified in nine hospitals and represented the major clone in four; and (iii) the Brazilian MRSA (17%)—PFGE C, ST239, spa type t037, SCC mec IIIA—was also detected in nine hospitals but never as the main clone. All isolates tested were PVL negative. Clone EMRSA-15 is currently the predominant MRSA clonal type circulating in Portuguese hospitals, but a new wave of MRSA has emerged in the country with the recent introduction and spread of the New York/Japan clone. The Brazilian MRSA that was the leading clone in Portugal in the late 1990s is declining and being progressively replaced by the two former clones. We report the first isolate SCC mec type V (ST45) in Portugal.

A clinical isolate of methicillin-resistant Staphylococcus aureus (MRSA) with intermediate resistance to vancomycin (minimal inhibitory concentration [MIC] of 4 mug/ml) was isolated in 2006 from a surgical wound of a patient hospitalized at the orthopedics ward of Hospital de São Marcos--Braga, in the town of Braga. A combination of molecular typing methods, including pulsed-field gel electrophoresis (PFGE), spa typing, multilocus sequence typing, and staphylococcal chromosomal cassette mec typing, identified the vancomycin intermediate-resistant S. aureus VISA-BRAGA as a derivative of the epidemic MRSA (EMRSA)-15 clone, which has been isolated with increasing frequency from several Portuguese hospitals recently. Compared to another EMRSA-15 isolate with the same genetic background (including PFGE subtype) the VISA-BRAGA isolate exhibited relatively high oxacillin MIC, slow growth, loss of hemolytic activity, and increased resistance to vancomycin and to daptomycin although neither of these two antibiotics was used in therapy. The VISA-BRAGA isolate described here appears to represent the first S. aureus with decreased susceptibility to vancomycin identified in a Portuguese hospital.

The prevalence of methicillin-resistant Staphylococcus aureus (MRSA) in Hungary has been increasing and is now close to 20% among invasive isolates of S. aureus. In order to understand the evolution of MRSA in Hungary, two collections of isolates were studied: 22 representatives of a collection of 238 MRSA isolates recovered between 1994 and 1998, and a collection of 299 MRSA isolates recovered between 2001 and 2004. The isolates were first characterised by pulsed-field gel electrophoresis (PFGE) and were distributed into 19 different PFGE patterns. Representatives of each pattern were further characterised by spa typing, multilocus sequence typing (MLST) and staphylococcal cassette chromosome mec (SCCmec) typing. The Hungarian clone that was predominant in 1994–1998 (PFGE E, ST239-III) had almost disappeared in 2003–2004, being replaced by the Southern German clone (PFGE B, ST228-I) and the New York/Japan epidemic clone (PFGE A, ST5-II), which represented c. 85% of the 2001–2004 isolates. Thus, this study describes, for the first time, the co-dominance and extensive spread of the New York/Japan clone in a European country.

Although the nosocomial prevalence of methicillin-resistant Staphylococcus aureus (MRSA) in Angola is over 60% and one of the highest in Africa, the extent of MRSA in the community is unknown. To fill this gap, we conducted a hospital-based study in which 158 children attending the emergency ward and ambulatory services of a pediatric hospital in Luanda, the capital of Angola, were screened for S. aureus nasal colonization. Overall, 70 (44.3%) individuals were colonized with S. aureus, of which 20 (28.6%) carried MRSA, resulting in a prevalence of 12.7% (20/158) of MRSA in the population screened. Molecular characterization by pulsed-field gel electrophoresis (PFGE), spa typing, multilocus sequence typing, and SCCmec typing distributed the isolates into two major MRSA clones and one dominant methicillin-susceptible S. aureus (MSSA) lineage, corresponding to the main clones circulating in hospitals in Luanda. The MRSA isolates mainly belonged to clones A (PFGE type A, spa type t105, ST5-IVa-65%) and B (PFGE B, t3869, ST88-IVa-30%), while MSSA isolates mainly belonged to clone L (PFGE type L, t861, ST508-42%). S. aureus isolates showed resistance to penicillin (96%), rifampin (87%), and trimethoprim-sulfamethoxazole (21%). In conclusion, the prevalence of MRSA among children in the community in Luanda is high and seems to originate from hospitals, warranting continuous monitoring and implementation of additional infection control measures.

Oxacillin-susceptible mecA-positive Staphylococcus aureus (OS-MRSA) isolates have been increasingly reported worldwide, but data regarding the African continent have not been available.Between 2010 and 2014, 1462 inpatients and healthcare workers were screened for MRSA nasal carriage in São Tomé and Príncipe (STP) and Angola, two Portuguese-speaking African countries (PALOP countries). We determined the presence of the mecA gene and the antimicrobial susceptibility profiles of the isolates. OS-MRSA clonal lineages were identified as well as the presence of virulence determinants, including Panton-Valentine leucocidin (PVL).Out of 164 S. aureus hospital isolates tested, 29 (17.7%) were mecA positive, but susceptible to oxacillin, showing oxacillin MICs ≤3 mg/L. All OS-MRSA isolates were resistant to cefoxitin and most of them were also resistant to at least two antimicrobials other than β-lactams. The 29 OS-MRSA were distributed into two major clonal lineages: (i) PFGE type B-ST88-SCCmec IVa, associated with spa types t186/t325/t786/t1814/t1951, detected in Angola (n = 5) and STP (n = 10); and (ii) PFGE type C-t451/t648-ST8-SCCmec V, exclusively found in STP (n = 9). OS-MRSA showed at least two virulence determinants. PVL was detected in an isolate recovered in STP.We describe a high prevalence of OS-MRSA among S. aureus strains recovered in two African countries. OS-MRSA in PALOP countries were mainly associated with ST88 and ST8, two prevalent MRSA clonal types in these countries. If direct testing for mecA is not available, cefoxitin susceptibility testing is highly recommended to avoid the misidentification of OS-MRSA.

Methicillin-resistant Staphylococcus aureus (MRSA) isolates ( n = 216), collected between January 1999 and May 2003 in a tertiary-care university hospital in Guadalajara, Mexico, were characterised by antibiotype, pulsed-field gel electrophoresis (PFGE) of Sma I macrorestriction fragments, and hybridisation of Cla I digests with mecA - and Tn 554 -specific DNA probes. Representatives of the single clonal type found were analysed by spa typing, multilocus sequence typing and staphylococcal chromosomal cassette mec (SCC mec ) typing, and were tested for the presence of 22 virulence determinants and agr type. A single PFGE pattern was identified, with minor variations over time, with spa type 2, sequence type 5, SCC mec type II, agr type 2 and the presence of the enterotoxin genes seg and sei , the γ-haemolysin variant gene hlg-v and the leukocidin lukE–lukD genes. In addition, the isolates showed antimicrobial resistance to β-lactams, macrolides, chloramphenicol and imipenem, and susceptibility to gentamicin, rifampicin, trimethoprim–sulphamethoxazole and vancomycin. Following its appearance in 1997, this clone spread within the hospital, and is now present in most of the hospital units and wards.

Methicillin-resistant Staphylococcus aureus (MRSA) emerged in the early 1960s after the acquisition of the methicillin resistance gene mecA, which is carried by the staphylococcal cassette chromosome mec (SCCmec). MRSA seemed to have arisen by multiple introductions of SCCmec into successful methicillin-susceptible S. aureus (MSSA) lineages. MRSA is one of the most common agents of nosocomial infections worldwide increasing the cost and mortality compared to MSSA infections. Little by little, MRSA has acquired resistance to all antibiotics available in clinical practice, which complicates treatment. This situation was further aggravated by the recent reports of vanA-mediated vancomycin-resistant S. aureus. As a reaction to the emergence and spread of multidrug-resistant MRSA worldwide, international surveillance systems such as the CEM/NET initiative have been created. The characterization of over 3000 MRSA isolates from different regions of the world evidenced the existence of only a few epidemic clones spread worldwide, namely the Iberian, Brazilian, Hungarian, New York/Japan, Pediatric and EMRSA-16 clones. It was found that in surveillance or evolutionary studies strains should be characterized by a combination of different typing methods, namely pulsed-field gel electrophoresis, multi-locus sequence typing and SCCmec typing. In recent years, community-acquired MRSA (CA-MRSA) has become a growing public health concern. However, although many authors reported the emergence of CA-MRSA isolates, a standard definition has not been created and the prevalence of MRSA among persons without risk factors seems to remain very low. CA-MRSA has distinct properties compared to epidemic nosocomial MRSA clones and its origin is still unclear. Certain authors suggest there is MRSA transmission from the hospital setting to the community, namely transfer of nosocomial MRSA minor clones or sporadic isolates showing a high degree of similarity with CA-MRSA; others believe CA-MRSA strains represent new acquisitions of SCCmec DNA in susceptible backgrounds. Many questions concerning this extraordinarily versatile and threatening pathogen remain unanswered, needing future investigation.