Epidemiology and clinical presentation of community-acquired Staphylococcus aureus bacteraemia in children under 5 years of age admitted to the Manhiça District Hospital, Mozambique, 2001-2019.

Staphylococcus aureus bacteraemia (SAB) is one of the most common bloodstream infections globally. Data on the burden and epidemiology of community-acquired SAB in low-income countries are scarce but needed to define preventive and management strategies. Blood samples were collected from children < 5 years of age with fever or severe disease admitted to the Manhiça District Hospital for bacterial isolation, including S. aureus. Between 2001 and 2019, 7.6% (3,197/41,891) of children had bacteraemia, of which 12.3% corresponded to SAB. The overall incidence of SAB was 56.1 episodes/100,000 children-years at risk (CYAR), being highest among neonates (589.8 episodes/100,000 CYAR). SAB declined significantly between 2001 and 2019 (322.1 to 12.5 episodes/100,000 CYAR). In-hospital mortality by SAB was 9.3% (31/332), and significantly associated with infections by multidrug-resistant (MDR) strains (14.7%, 11/75 vs. 6.9%, 14/204 among non-MDR, p = 0.043) and methicillin-resistant S. aureus (33.3%, 5/15 vs. 7.6%, 20/264 among methicillin-susceptible S. aureus, p = 0.006). Despite the declining rates of SAB, this disease remains an important cause of death among children admitted to MDH, possibly in relation to the resistance to the first line of empirical treatment in use in our setting, suggesting an urgent need to review current policy recommendations.

Occurrence and Variability of the Efflux Pump Gene norA across the Staphylococcus Genus.

NorA is one of the main native MDR efflux pumps of Staphylococcus aureus, contributing to reduced susceptibility towards fluoroquinolones and biocides, but little is known about its variability within S. aureus or its distribution and conservation among other staphylococci. We screened for sequences homologous to S. aureus norA and found it in 61 out of the 63 Staphylococcus species described. To the best of our knowledge, this is the first study to report the occurrence of norA across the Staphylococcus genus. The norA phylogenetic tree follows the evolutionary relations of staphylococci and the closely related Mammalliicoccus genus. Comparative analyses suggest a conservation of the NorA function in staphylococci. We also analyzed the variability of norA within S. aureus, for which there are several circulating norA alleles, differing up to 10% at the nucleotide level, which may hamper proper norA detection. We demonstrate the applicability of a PCR-based algorithm to detect and differentiate norA alleles in 52 S. aureus representing a wider collection of 89 isolates from different hosts. Our results highlight the prevalence of norAI and norAII in different settings and the association of norA alleles with specific S. aureus clonal lineages. Ultimately, it confirms the applicability of our PCR-based algorithm to rapidly detect and assign the different norA alleles, a trait that may impact antimicrobial efflux capacity and the search for potential NorA inhibitors.

Active antimicrobial efflux in Staphylococcus epidermidis: building up of resistance to fluoroquinolones and biocides in a major opportunistic pathogen.

Objectives: To analyse the efflux-mediated response of Staphylococcus epidermidis to ethidium bromide (EtBr), a substrate of multidrug efflux pumps (EPs). Methods: The susceptible reference strain S. epidermidis ATCC 12228 was exposed to a step-wise adaptation to EtBr. The resulting EtBr-adapted strains were characterized regarding their antibiotic and biocide susceptibility by MIC determination and evaluation of efflux activity by re-determination of MICs in the presence of known efflux inhibitors and real-time fluorometry. Mutations in the QRDR of grlA and gyrA were screened by sequencing. The expression levels of S. epidermidis homologues of the main Staphylococcus aureus EP genes were quantified by RT-qPCR. Results: Exposure to EtBr led to a gradual increase in resistance to antimicrobials, with the final EtBr-adapted strain, ATCC 12228_EtBr, displaying phenotypic resistance to fluoroquinolones and reduced susceptibility to several antiseptics and disinfectants, although no mutations were detected in the QRDR of the grlA/gyrA genes. A reduction in the MICs of fluoroquinolones and selected biocides promoted by efflux inhibitors suggested an efflux-mediated response to EtBr exposure. Detailed analysis of the EtBr-adapted strains detected a gradual increase in efflux activity. Gene expression assays revealed a temporal activation of S. epidermidis EPs, with an early response involving norA, SE2010 and SE1103 followed by a late response mediated by norA, which coincided with the occurrence of the mutation -1A→T in the norA promoter region. Conclusions: This study demonstrated that S. epidermidis has the potential to develop a multiple resistance phenotype mediated by efflux when exposed to a non-antibiotic substrate of multidrug EPs.

Exploring the virulence potential of Staphylococcus aureus CC121 and CC152 lineages related to paediatric community-acquired bacteraemia in Manhiça, Mozambique.

Staphylococcus aureus is a frequent agent of bacteraemia. This bacterium has a variety of virulence traits that allow the establishment and maintenance of infection. This study explored the virulence profile of S. aureus strains causing paediatric bacteraemia (SAB) in Manhiça district, Mozambique. We analysed 336 S. aureus strains isolated from blood cultures of children younger than 5 years admitted to the Manhiça District Hospital between 2001 and 2019, previously characterized for antibiotic susceptibility and clonality. The strains virulence potential was evaluated by PCR detection of the Panton-Valentine leucocidin (PVL) encoding genes, lukS-PV/lukF-PV, assessment of the capacity for biofilm formation and pathogenicity assays in Galleria mellonella. The overall carriage of PVL-encoding genes was over 40%, although reaching ~ 70 to 100% in the last years (2014 to 2019), potentially linked to the emergence of CC152 lineage. Strong biofilm production was a frequent trait of CC152 strains. Representative CC152 and CC121 strains showed higher virulence potential in the G. mellonella model when compared to reference strains, with variations within and between CCs. Our results highlight the importance of monitoring the emergent CC152-MSSA-PVL+ and other lineages, as they display important virulence traits that may negatively impact the management of SAB paediatric patients in Manhiça district, Mozambique.

Exploring Efflux as a Mechanism of Reduced Susceptibility towards Biocides and Fluoroquinolones in Staphylococcus pseudintermedius.

Staphylococcus pseudintermedius is the main bacterial cause of skin and soft tissue infections (SSTIs) in companion animals, particularly dogs. The emergence of methicillin-resistant S. pseudintermedius (MRSP) strains, frequently with multidrug resistance phenotypes is a public health concern. This study aimed to evaluate efflux, a resistance mechanism still poorly characterized in S. pseudintermedius, as a contributor to biocide and fluoroquinolone resistance. Susceptibility to the efflux pump substrates ethidium bromide (EtBr), tetraphenylphosphonium bromide (TPP) and ciprofloxacin (CIP) was evaluated by minimum inhibitory concentration (MIC) determination for 155 SSTIs-related S. pseudintermedius in companion animals. EtBr and TPP MIC distributions were analyzed to estimate cut-off (COWT) values. The effect of the efflux inhibitors (EIs) thioridazine and verapamil was assessed upon MICs and fluorometric EtBr accumulation assays, performed with/without glucose and/or EIs. This approach detected a non-wild type population towards TPP with increased efflux, showed to be strain-specific and glucose-dependent. Resistance to fluoroquinolones was mainly linked to target gene mutations, yet a contribution of efflux on CIP resistance levels could not be ruled out. In sum, this study highlights the relevance of efflux-mediated resistance in clinical S. pseudintermedius, particularly to biocides, and provides a methodological basis for further studies on the efflux activity on this important pathogen of companion animals.

Genetic diversity and antimicrobial resistance profiles of Staphylococcus pseudintermedius associated with skin and soft-tissue infections in companion animals in Lisbon, Portugal.

Staphylococcus pseudintermedius is the main bacterial pathogen of skin and soft-tissue infections (SSTIs) in companion animals. Antimicrobial resistance in this species is a growing public health concern. This study aims to characterize a collection of S. pseudintermedius causing SSTIs in companion animals, establishing the main clonal lineages and antimicrobial resistance traits. The collection corresponded to all S. pseudintermedius (n = 155) causing SSTIs in companion animals (dogs, cats and one rabbit) collected between 2014 and 2018 at two laboratories in Lisbon, Portugal. Susceptibility patterns were established by disk diffusion for 28 antimicrobials (15 classes). For antimicrobials without clinical breakpoints available, a cut-off value (COWT) was estimated, based on the distribution of the zones of inhibition. The blaZ and mecA genes were screened for the entire collection. Other resistance genes (e.g., erm, tet, aadD, vga(C), dfrA(S1)) were searched only for those isolates showing an intermediate/resistance phenotype. For fluoroquinolone resistance, we determined the chromosomal mutations in the target genes grlA and gyrA. All the isolates were typed by PFGE following SmaI macrorestriction and isolates representative of each PFGE type were further typed by MLST. Forty-eight out of the 155 S. pseudintermedius isolates (31.0%) were methicillin-resistant (mecA +, MRSP). Multidrug-resistant (MDR) phenotypes were detected for 95.8% of the MRSP and 22.4% of the methicillin-susceptible (MSSP) isolates. Of particular concern, only 19 isolates (12.3%) were susceptible to all antimicrobials tested. In total, 43 different antimicrobial resistance profiles were detected, mostly associated with the carriage of blaZ, mecA, erm(B), aph3-IIIa, aacA-aphD, cat pC221, tet(M) and dfr(G) genes. The 155 isolates were distributed within 129 PFGE clusters, grouped by MLST in 42 clonal lineages, 25 of which correspond to new sequence types (STs). While ST71 remains the most frequent S. pseudintermedius lineage, other lineages that have been replacing ST71 in other countries were detected, including ST258, described for the first time in Portugal. This study revealed a high frequency of MRSP and MDR profiles among S. pseudintermedius associated with SSTIs in companion animals in our setting. Additionally, several clonal lineages with different resistance profiles were described, evidencing the importance of a correct diagnosis and selection of the therapy.

Antimicrobial resistance and clonality of Staphylococcus aureus causing bacteraemia in children admitted to the Manhiça District Hospital, Mozambique, over two decades.

Background: Staphylococcus aureus is one of the main causes of bacteraemia, associated with high mortality, mainly due to the occurrence of multidrug resistant (MDR) strains. Data on antibiotic susceptibility and genetic lineages of bacteraemic S. aureus are still scarce in Mozambique. The study aims to describe the antibiotic susceptibility and clonality of S. aureus isolated from blood cultures of children admitted to the Manhiça District Hospital over two decades (2001-2019). Methods: A total of 336 S. aureus isolates detected in blood cultures of children aged <5 years were analyzed for antibiotic susceptibility by disk diffusion or minimal inhibitory concentration, and for the presence of resistance determinants by PCR. The clonality was evaluated by SmaI-PFGE, spa typing, and MLST. The SCCmec element was characterized by SCCmec typing. Results: Most S. aureus (94%, 317/336) were resistant to at least one class of antibiotics, and one quarter (25%) showed a MDR phenotype. High rates of resistance were detected to penicillin (90%) and tetracycline (48%); followed by erythromycin/clindamycin (25%/23%), and co-trimoxazole (11%), while resistance to methicillin (MRSA strains) or gentamicin was less frequent (≤5%). The phenotypic resistance to distinct antibiotics correlated well with the corresponding resistance determinants (Cohen's κ test: 0.7-1.0). Molecular typing revealed highly diverse clones with predominance of CC5 (17%, 58/336) and CC8 (16%), followed by CC15 (11%) and CC1 (11%). The CC152, initially detected in 2001, re-emerged in 2010 and became predominant throughout the remaining surveillance period, while other CCs (CC1, CC5, CC8, CC15, CC25, CC80, and CC88) decreased over time. The 16 MRSA strains detected belonged to clones t064-ST612/CC8-SCCmecIVd (69%, 11/16), t008-ST8/CC8-SCCmecNT (25%, 4/16) and t5351-ST88/CC88-SCCmecIVa (6%, 1/16). Specific clonal lineages were associated with extended length of stay and high in-hospital mortality. Conclusion: We document the circulation of diverse MDR S. aureus causing paediatric bacteraemia in Manhiça district, Mozambique, requiring a prompt recognition of S. aureus bacteraemia by drug resistant clones to allow more targeted clinical management of patients.

Staphylococcus aureus Causing Skin and Soft Tissue Infections in Companion Animals: Antimicrobial Resistance Profiles and Clonal Lineages.

Staphylococcus aureus is a relevant agent of skin and soft tissue infections (SSTIs) in animals. Fifty-five S. aureus comprising all SSTI-related isolates in companion animals, collected between 1999 and 2018 (Lab 1) or 2017 and 2018 (Lab 2), were characterized regarding susceptibility to antibiotics and heavy metals and carriage of antimicrobial resistance determinants. Clonal lineages were established by PFGE, MLST and agr typing. Over half of the isolates (56.4%, 31/55) were methicillin-resistant S. aureus (MRSA), and 14.5% showed a multidrug resistance (MDR) phenotype. Resistance was most frequently observed for beta-lactams (81.8%, related to blaZ and/or mecA), fluoroquinolones (56.4%) and macrolides/lincosamides (14.5%, related to erm(A) or erm(C)). The distributions of heavy-metal MICs allowed the detection of non-wild-type populations associated with several resistance genes. The collection showed genetic diversity, with prevalence of clonal lineage ST22-agrI (45.5%, 25/55), comprising only MRSA isolates, and several less frequently detected clones, including ST5-agrII (14.6%, 8/55), ST398-agrI (9.1%, 5/55) and ST72-agrI (7.3%, 4/55). This work highlights the high frequency of SSTI-related MRSA strains that reflect the clonal lineages circulating both in companion animals and humans in Portugal, reinforcing the need for a One Health approach when studying staphylococci causing infections in companion animals.

Virulence Potential of Biofilm-Producing Staphylococcus pseudintermedius, Staphylococcus aureus and Staphylococcus coagulans Causing Skin Infections in Companion Animals.

Coagulase-positive staphylococci (CoPS) account for most bacteria-related pyoderma in companion animals. Emergence of methicillin-resistant strains of Staphylococcus pseudintermedius (MRSP), Staphylococcus aureus (MRSA) or Staphylococcus coagulans (MRSC), often with multidrug-resistant (MDR) phenotypes, is a public health concern. The study collection comprised 237 staphylococci (S. pseudintermedius (n = 155), S. aureus (n = 55) and S. coagulans (n = 27)) collected from companion animals, previously characterized regarding resistance patterns and clonal lineages. Biofilm production was detected for 51.0% (79/155), 94.6% (52/55) and 88.9% (24/27) of the S. pseudintermedius, S. aureus and S. coagulans, respectively, and was a frequent trait of the predominant S. pseudintermedius and S. aureus clonal lineages. The production of biofilm varied with NaCl supplementation of the growth media. All S. pseudintermedius and S. aureus strains carried icaADB. Kaplan-Meier survival analysis of Galleria mellonella infected with different CoPS revealed a higher virulence potential of S. aureus when compared with other CoPS. Our study highlights a high frequency of biofilm production by prevalent antimicrobial-resistant clonal lineages of CoPS associated with animal pyoderma, potentially related with a higher virulence potential and persistent or recurrent infections.

Exploring the contribution of efflux on the resistance to fluoroquinolones in clinical isolates of Staphylococcus aureus.

BACKGROUND: Antimicrobial resistance mediated by efflux systems is still poorly characterized in Staphylococcus aureus, despite the description of several efflux pumps (EPs) for this bacterium. In this work we used several methodologies to characterize the efflux activity of 52 S. aureus isolates resistant to ciprofloxacin collected in a hospital in Lisbon, Portugal, in order to understand the role played by these systems in the resistance to fluoroquinolones. RESULTS: Augmented efflux activity was detected in 12 out of 52 isolates and correlated with increased resistance to fluoroquinolones. Addition of efflux inhibitors did not result in the full reversion of the fluoroquinolone resistance phenotype, yet it implied a significant decrease in the resistance levels, regardless of the type(s) of mutation(s) found in the quinolone-resistance determining region of grlA and gyrA genes, which accounted for the remaining resistance that was not efflux-mediated. Expression analysis of the genes coding for the main efflux pumps revealed increased expression only in the presence of inducing agents. Moreover, it showed that not only different substrates can trigger expression of different EP genes, but also that the same substrate can promote a variable response, according to its concentration. We also found isolates belonging to the same clonal type that showed different responses towards drug exposure, thus evidencing that highly related clinical isolates may diverge in the efflux-mediated response to noxious agents. The data gathered by real-time fluorometric and RT-qPCR assays suggest that S. aureus clinical isolates may be primed to efflux antimicrobial compounds. CONCLUSIONS: The results obtained in this work do not exclude the importance of mutations in resistance to fluoroquinolones in S. aureus, yet they underline the contribution of efflux systems for the emergence of high-level resistance. All together, the results presented in this study show the potential role played by efflux systems in the development of resistance to fluoroquinolones in clinical isolates of S. aureus.

Phenotypic and Molecular Traits of Staphylococcus coagulans Associated with Canine Skin Infections in Portugal.

Staphylococcus coagulans is among the three most frequent pathogens of canine pyoderma. Yet, studies on this species are scarce. Twenty-seven S. coagulans and one S. schleiferi, corresponding to all pyoderma-related isolations from these two species at two veterinary laboratories in Lisbon, Portugal, between 1999 and 2018 (Lab 1) or 2018 (Lab 2), were analyzed. Isolates were identified by the analysis of the nuc gene and urease production. Antibiotic susceptibility towards 27 antibiotics was evaluated by disk diffusion. Fourteen antibiotic resistance genes were screened by PCR. Isolates were typed by SmaI-PFGE. Two S. coagulans isolates (2/27, 7.4%) were methicillin-resistant (MRSC, mecA+) and four (4/27, 14.8%) displayed a multidrug-resistant (MDR) phenotype. We observed resistance to penicillin (17/27, 63.0%), fluoroquinolones (11/27, 40.7%), erythromycin and clindamycin (3/27, 11.1%), fusidic acid (3/27, 11.1%) and tetracycline (1/27, 3.7%). The blaZ and erm(B) genes were carried by 16 and 1 isolates resistant to penicillin and erythromycin/clindamycin, respectively. Only three S. coagulans carried plasmids. The single S. schleiferi isolate presented an MDR phenotype. SmaI-PFGE revealed a limited genetic diversity of S. coagulans, with a predominant lineage present from 2001 to 2018. This study describes the first MRSC causing canine infection in Portugal and reveals a high burden of antimicrobial resistance, with the emergence of MDR phenotypes within the main lineages.

Clonal Lineages, Antimicrobial Resistance, and PVL Carriage of Staphylococcus aureus Associated to Skin and Soft-Tissue Infections from Ambulatory Patients in Portugal.

Staphylococcus aureus (S. aureus) is a leading cause of skin and soft-tissue infections (SSTIs) in the community. In this study, we characterized a collection of 34 S. aureus from SSTIs in ambulatory patients in Portugal and analyzed the presence of Panton-Valentine leucocidin (PVL)-encoding genes and antibiotic-resistance profile, which was correlated with genetic determinants, plasmid carriage, and clonal lineage. Nearly half of the isolates (15, 44.1%) were methicillin-resistant Staphylococcus aureus (MRSA) and/or multidrug resistant (MDR). We also detected resistance to penicillin (33/34, 97.1%), fluoroquinolones (17/34, 50.0%), macrolides and lincosamides (15/34, 44.1%), aminoglycosides (6/34, 17.6%), and fusidic acid (2/34, 5.9%), associated with several combinations of resistance determinants (blaZ, erm(A), erm(C), msr(A), mph(C), aacA-aphD, aadD, aph(3')-IIIa, fusC), or mutations in target genes (fusA, grlA/gyrA). The collection presented a high genetic diversity (Simpson's index of 0.92) with prevalence of clonal lineages CC5, CC22, and CC8, which included the MRSA and also most MDR isolates (CC5 and CC22). PVL-encoding genes were found in seven isolates (20.6%), three methicillin-susceptible Staphylococcus aureus (MSSA) (ST152-agrI and ST30-agrIII), and four MRSA (ST8-agrI). Plasmid profiling revealed seventeen distinct plasmid profiles. This work highlights the high frequency of antimicrobial resistance and PVL carriage in SSTIs-related S. aureus outside of the hospital environment.

Proposal of Epidemiological Cutoff Values for Apramycin 15 µg and Florfenicol 30 µg Disks Applicable to Staphylococcus aureus.

Apramycin and florfenicol are two antimicrobial agents exclusively used in veterinary medicine. Resistance determinants to these antimicrobial agents have been described in several staphylococci, yet no inhibition zone-based epidemiological cutoff (ECOFF) values are available to detect populations harboring resistance mechanisms. In this study, we propose disk diffusion inhibition zone ECOFF values of Staphylococcus aureus for apramycin and florfenicol. The susceptibility to apramycin and florfenicol was evaluated by disk diffusion of five S. aureus collections, comprising 352 isolates of animal (n = 265) and human (n = 87) origin. The aggregated distributions of inhibition zone diameters were analyzed by the normalized resistance interpretation method to obtain normalized wild-type (WT) population distributions and corresponding ECOFF values. The putative WT populations of S. aureus were characterized by an inhibition zone ≥15 mm (ECOFF = 15 mm) for apramycin and ≥21 mm for florfenicol (ECOFF = 21 mm). Five nonwild-type (NWT) isolates were detected for apramycin, all without inhibition zone and harboring the apmA gene, whereas five NWT isolates were identified for florfenicol, all carrying the fexA gene. The proposed ECOFF values for apramycin and florfenicol may be a valuable tool in future antimicrobial resistance monitoring and surveillance studies to identify S. aureus NWT populations toward these antimicrobial agents.

Characterization of intrinsic efflux activity of Enterococcus faecalis ATCC29212 by a semi-automated ethidium bromide method.

Enterococcus faecalis is recognized as a multidrug-resistant nosocomial pathogen. The phenotypic basis for this is largely uncharacterized. The intrinsic efflux system of the antibiotic-susceptible E. faecalis ATCC29212 strain was studied using a semi-automated method that assesses accumulation and efflux of the universal efflux pump substrate ethidium bromide (EB). The results show that the intrinsic efflux system of this Enterococcus strain is controlled by energy derived from the catabolism of glucose and the proton concentration of the medium. At pH 5, agents that inhibit efflux pumps in Gram-positive organisms and the proton gradient un-coupler CCCP do not increase accumulation nor inhibit efflux of EB. In contrast, at pH 8, where the proton concentration is 1,000-fold lower, these agents increase accumulation and efflux of EB. These results are relevant to infections produced by E. faecalis and subsequent antibiotic therapy with antibiotics to which the organism is known to be intrinsically resistant.

Efflux-mediated response of Staphylococcus aureus exposed to ethidium bromide.

OBJECTIVES: By adapting an antibiotic-susceptible Staphylococcus aureus strain to increasing concentrations of ethidium bromide, a known substrate of efflux pumps (EPs), and by phenotypically and genotypically analysing the resulting progeny, we characterized the molecular mechanisms of S. aureus adaptation to ethidium bromide. METHODS: S. aureus ATCC 25923 was grown in increasing concentrations of ethidium bromide. The MICs of representatives of eight classes of antibiotics, eight biocides and two dyes against ATCC 25923 and its ethidium bromide-resistant progeny ATCC 25923(EtBr) were determined with or without six efflux pump inhibitors (EPIs). Efflux activity in the presence/absence of EPIs was evaluated by real-time fluorometry. The presence and expression of eight EP genes were assayed by PCR and quantitative RT-PCR (qRT-PCR), respectively. Mutations in grlA, gyrA and norA promoter regions were screened by DNA sequencing. RESULTS: Compared with its parental strain, ATCC 25923(EtBr) was 32-fold more resistant to ethidium bromide and also more resistant to biocides and hydrophilic fluoroquinolones. Resistance to these could be reduced by the EPIs chlorpromazine, thioridazine and reserpine. Increased efflux of ethidium bromide by ATCC 25923(EtBr) could be inhibited by the same EPIs. qRT-PCR showed that norA was 35-fold over-expressed in ATCC 25923(EtBr), whereas the remaining EP genes showed no significant increase in their expression. Sequencing of the norA promoter region revealed a 70 bp deletion in ATCC 25923(EtBr). CONCLUSIONS: Exposure of S. aureus to quaternary compounds such as ethidium bromide results in decreased susceptibility of the organism to a wide variety of compounds, including quinolones and biocides through an efflux-mediated response, which for strain ATCC 25923 is mainly NorA-mediated. This altered expression may result from alterations in the norA promoter region.

First report on antimicrobial resistance and molecular characterisation of Salmonella enterica serotype Typhi isolated from human specimens in Luanda, Angola.

OBJECTIVES: Typhoid fever is a common infection in Africa and, despite scarce surveillance reports, its incidence is commonly considered high by the Angolan health system. Drug-resistant Salmonella enterica serotype Typhi has emerged, making antimicrobial susceptibility testing essential to provide clinical guidance. This is the first report analysing the antimicrobial resistance patterns and population structure of the few S. enterica ser. Typhi isolated from patients with typhoid fever in Luanda, Angola. METHODS: Isolates were collected by the Angolan National Institute of Public Health between September 2013 and May 2014. Ten isolates were identified by the API 20E system and serotyping, and the genus was confirmed by PCR. All isolates were tested for antibiotic susceptibility, and the presence of resistance genes [blaCTX-M, blaSHV, blaTEM, blaOXA-1, several plasmid-borne genes encoding AmpC ß-lactamases, sul and qnr genes, dfrIa, dfrA12, aac(6')-Ib, cmlA and floR] were screened by PCR. Isolates were typed by PFGE and MLST. RESULTS: Several isolates were identified with resistance to trimethoprim/sulfamethoxazole (n=6), ß-lactams (n=5) and chloramphenicol (n=1) and reduced susceptibility to ciprofloxacin (n=2). PFGE revealed eight closely related restriction patterns, and MLST grouped these into three sequence types (ST1, ST2 and ST8), with ST2 being predominant. CONCLUSION: This first epidemiological report provides a preliminary description of S. enterica ser. Typhi strains circulating in Luanda and emphasises the need for continuous monitoring of this pathogen to provide information in order to implement better epidemiological strategies for the control of typhoid fever in Angola.

Genetic Diversity of norA, Coding for a Main Efflux Pump of Staphylococcus aureus.

NorA is the best studied efflux system of Staphylococcus aureus and therefore frequently used as a model for investigating efflux-mediated resistance in this pathogen. NorA activity is associated with resistance to fluoroquinolones, several antiseptics and disinfectants and several reports have pointed out the role of efflux systems, including NorA, as a first-line response to antimicrobials in S. aureus. Genetic diversity studies of the gene norA have described three alleles; norAI, norAII and norAIII. However, the epidemiology of these alleles and their impact on NorA activity remains unclear. Additionally, increasing studies do not account for norA variability when establishing relations between resistance phenotypes and norA presence or reported absence, which actually corresponds, as we now demonstrate, to different norA alleles. In the present study we assessed the variability of the norA gene present in the genome of over 1,000 S. aureus isolates, corresponding to 112 S. aureus strains with whole genome sequences publicly available; 917 MRSA strains sourced from a London-based study and nine MRSA isolates collected in a major Hospital in Lisbon, Portugal. Our analyses show that norA is part of the core genome of S. aureus. It also suggests that occurrence of norA variants reflects the population structure of this major pathogen. Overall, this work highlights the ubiquitous nature of norA in S. aureus which must be taken into account when studying the role played by this important determinant on S. aureus resistance to antimicrobials.

In vitro and in silico analysis of the efficiency of tetrahydropyridines as drug efflux inhibitors in Escherichia coli.

The objectives of this study were to evaluate tetrahydropyridine derivatives as efflux inhibitors and to understand the mechanism of action of the compounds by in silico studies. Minimum inhibitory concentration (MIC) determination, fluorometric methods and docking simulations were performed. The compounds NUNL02, NUNL09 and NUNL10 inhibited efflux, and NUNL02 is very likely a substrate of the transporter protein AcrB. Docking studies suggested that the mechanism of action could be by competition with substrate for binding sites and protein residues. We showed for the first time the potential of tetrahydropyridines as efflux inhibitors and highlighted compound NUNL02 as an AcrB-specific inhibitor. Docking studies suggested that competition is the putative mechanism of action of these compounds.

Plasmid-Borne Antimicrobial Resistance of Staphylococcus aureus Isolated in a Hospital in Lisbon, Portugal.

Plasmids play a key role in the genetic plasticity and survival of Staphylococcus aureus in challenging environments. Although many S. aureus plasmids have been described, still few studies portray the plasmid content of a given S. aureus population. The aim of this work was to characterize the plasmids carried by a collection of 53 S. aureus isolates collected in a large hospital in Lisbon, Portugal, and investigate their role in conferring resistance to several antimicrobial agents. Plasmids were present in 44 out of the 53 isolates and were grouped into eleven AccI restriction profiles. Plasmid curing of representative strains and comparison of antimicrobial susceptibility profiles between pairs of isogenic strains proved to be a valuable guidance tool in the identification of plasmid-located resistance genes. The plasmids harbored several resistance genes, namely blaZ (resistance to ß-lactams), erm(C) (resistance to macrolides, lincosamides, and streptogramin B), cadA (resistance to cadmium and zinc), cadD (resistance to cadmium), and qacA and smr (resistance to biocides and dyes). This study demonstrates the impact of plasmids on the resistance properties of S. aureus, highlighting their role in the dissemination of antibiotic, heavy metal, and biocide resistance genes, and survival of this major pathogen in the hospital environment.

An Experimental Model for the Rapid Screening of Compounds with Potential Use Against Mycobacteria.

Infections caused by Mycobacterium tuberculosis and other mycobacteria are major challenges for global public health. Particularly worrisome are infections caused by multidrug-resistant bacteria, which are increasingly difficult to treat because of the loss of efficacy of the current antibacterial agents, a problem that continues to escalate worldwide. There has been a limited interest and investment on the development of new antibacterial agents in the past decades. This has led to the current situation, in which there is an urgent demand for innovative therapeutic alternatives to fight infections caused by multidrug-resistant pathogens, such as multidrug-resistant tuberculosis. The identification of compounds that can act as adjuvants in antimycobacterial therapeutic regimens is an appealing strategy to restore the efficacy lost by some of the antibiotics currently used and shorten the duration of the therapeutic regimen. In this work, by setting Mycobacterium smegmatis as a model organism, we have developed a methodological strategy to identify, in a fast and simple approach, compounds with antimycobacterial activity or with potential adjuvant properties, by either inhibition of efflux or other unrelated mechanisms. Such an approach may increase the rate of identification of promising molecules, to be further explored in pathogenic models for their potential use either as antimicrobials or as adjuvants, in combination with available therapeutic regimens for the treatment of mycobacterial infections. This method allowed us to identify a new molecule that shows promising activity as an efflux inhibitor in M. smegmatis.