Search

Abstract Background Enterococci have emerged as the third most common cause of nosocomial infections, requiring bactericidal antimicrobial therapy. Although vancomycin resistance is a major problem in clinics and has emerged in an important extend in farm animals, few studies have examined it in wild animals. To determine the prevalence of vanA-containing Enterococcus strains among faecal samples of Seagulls (Larus cachinnans) of Berlengas Natural Reserve of Portugal, we developed a proteomic approach integrated with genomic data. The purpose was to detect the maximum number of proteins that vary in different enterococci species which are thought to be connected in some, as yet unknown, way to antibiotic resistance. Results From the 57 seagull samples, 54 faecal samples showed the presence of Enterococcus isolates (94.7%). For the enterococci, E. faecium was the most prevalent species in seagulls (50%), followed by E. faecalis and E. durans (10.4%), and E. hirae (6.3%). VanA-containing enterococcal strains were detected in 10.5% of the 57 seagull faecal samples studied. Four of the vanA-containing enterococci were identified as E. faecium and two as E. durans. The tet(M) gene was found in all five tetracycline-resistant vanA strains. The erm(B) gene was demonstrated in all six erythromycin-resistant vanA strains. The hyl virulence gene was detected in all four vanA-containing E. faecium isolates in this study, and two of them harboured the purK1 allele. In addition these strains also showed ampicillin and ciprofoxacin resistance. The whole-cell proteomic profile of vanA-containing Enterococcus strains was applied to evaluate the discriminatory power of this technique for their identification. The major differences among species-specific profiles were found in the positions corresponding to 97-45 kDa. Sixty individualized protein spots for each vanA isolate was identified and suitable for peptide mass fingerprinting measures by spectrometry measuring (MALDI/TOF MS) and their identification through bioinformatic databases query. The proteins were classified in different groups according to their biological function: protein biosynthesis, ATP synthesis, glycolysis, conjugation and antibiotic resistance. Taking into account the origin of these strains and its relation to infectious processes in humans and animals, it is important to explore the proteome of new strains which might serve as protein biomarkers for biological activity. Conclusions The comprehensive description of proteins isolated from vancomycin-resistant Enterococcus faecium and E. durans may provide new targets for development of antimicrobial agents. This knowledge may help to identify new biomarkers of antibiotic resistance and virulence factors.

Methicillin-resistant Staphylococcus aureus (MRSA) is a major human pathogen worldwide, and although surveillance studies are available in the most developed countries, data from Angola are inexistent. In June 2012, 295 inpatients and 199 healthcare workers from three hospitals in Luanda, Angola were nasal swabbed for S. aureus and MRSA carriage. A total of 117 individuals (23.7%) were S. aureus nasal carriers, out of which 68 (58.1%) were colonized with MRSA. The majority of the MRSA isolates (74%) belonged to a single clonal lineage, pulsed-field gel electrophoresis (PFGE) A-ST5-IVa associated with three spa types ( spa types t105/t311/t11657), followed by PFGE C-ST88-IVa ( spa types t186/t325/t786/t1951/t3869) ( n=9; 12%); the other 11 MRSA isolates were representatives of 4 additional lineages. Almost half (49%) of the methicillin-susceptible S. aureus (MSSA) isolates belonged to three major clones: PFGE B-ST508 ( spa types t050/t861/t1346/t1574/t2626/t12218), PFGE D-ST45 ( spa types t939/t11656), and PFGE E-ST30 ( spa types t1202/t9118). MSSA isolates presented a high variability of virulence factors, including Panton-Valentine leukocidine (7.9%). MRSA carriage in Luanda is considerably high, and the major clone corresponds to a worldwide epidemic lineage, so far scarcely reported in Africa. Additional infection control measures in this metropolis are mandatory for a global MRSA control. [ABSTRACT FROM AUTHOR]

Background: Enterococci have emerged as the third most common cause of nosocomial infections, requiring bactericidal antimicrobial therapy. Although vancomycin resistance is a major problem in clinics and has emerged in an important extend in farm animals, few studies have examined it in wild animals. To determine the prevalence of vanA-containing Enterococcus strains among faecal samples of Seagulls (Larus cachinnans) of Berlengas Natural Reserve of Portugal, we developed a proteomic approach integrated with genomic data. The purpose was to detect the maximum number of proteins that vary in different enterococci species which are thought to be connected in some, as yet unknown, way to antibiotic resistance. Results: From the 57 seagull samples, 54 faecal samples showed the presence of Enterococcus isolates (94.7%). For the enterococci, E. faecium was the most prevalent species in seagulls (50%), followed by E. faecalis and E. durans (10.4%), and E. hirae (6.3%). VanA-containing enterococcal strains were detected in 10.5% of the 57 seagull faecal samples studied. Four of the vanA-containing enterococci were identified as E. faecium and two as E. durans. The tet(M) gene was found in all five tetracycline-resistant vanA strains. The erm(B) gene was demonstrated in all six erythromycin-resistant vanA strains. The hyl virulence gene was detected in all four vanA-containing E. faecium isolates in this study, and two of them harboured the purK1 allele. In addition these strains also showed ampicillin and ciprofoxacin resistance. The whole-cell proteomic profile of vanA-containing Enterococcus strains was applied to evaluate the discriminatory power of this technique for their identification. The major differences among species-specific profiles were found in the positions corresponding to 97-45 kDa. Sixty individualized protein spots for each vanA isolate was identified and suitable for peptide mass fingerprinting measures by spectrometry measuring (MALDI/TOF MS) and their identification through bioinformatic databases query. The proteins were classified in different groups according to their biological function: protein biosynthesis, ATP synthesis, glycolysis, conjugation and antibiotic resistance. Taking into account the origin of these strains and its relation to infectious processes in humans and animals, it is important to explore the proteome of new strains which might serve as protein biomarkers for biological activity. Conclusions: The comprehensive description of proteins isolated from vancomycin-resistant Enterococcus faecium and E. durans may provide new targets for development of antimicrobial agents. This knowledge may help to identify new biomarkers of antibiotic resistance and virulence factors. [ABSTRACT FROM AUTHOR]

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., (Copyright © 2015, American Society for Microbiology. All Rights Reserved.)

Fifty-four healthy dogs were screened in Portugal for the presence of nasal methicillin-resistant Staphylococcus aureus (MRSA) carriage. Sixteen MRSA isolates (one/sample) were recovered from nasal samples of dogs, and they were typed by molecular methods (S. aureus protein A [spa]-, multilocus sequence typing-, staphylococcal cassette chromosome mec-typing). MRSA isolates were investigated for their susceptibility to antimicrobial agents by disk-diffusion test. The presence of resistance genes and of the Panton-Valentine leukocidin gene (lukF-lukS) was analyzed by PCR. Four different spa-types were identified among our MRSA isolates (t032, t432, t747, and t4726), with t032 as the most frequently detected. The sequence-type ST22 was identified in four tested MRSA isolates with different spa-types. All 16 isolates presented the staphylococcal cassette chromosome mec type IV. Most of MRSA isolates were resistant to ciprofloxacin, erythromycin, and clindamycin (94%-100%), and no resistance was identified to chloramphenicol, mupirocin, and trimethoprim-sulfametoxazole. The ermC and tetM resistance genes were detected in all MRSA isolates. The amino acid changes Ser84Leu in GyrA protein and Ser80Phe in GrlA protein were the most prevalent ones in our MRSA isolates. None of the MRSA strains carried the lukF-lukS genes. The results presented in this study indicate that healthy dogs may be a reservoir of MRSA that could be transmitted to humans by direct contact.

There is a great concern by the emergence and the wide dissemination of extended-spectrum beta-lactamases (ESBLs) among animal Escherichia coli isolates. We intended to determinate the carriage level and type of ESBLs in E. coli obtained from fecal samples from pigs raised on an intensive pig farm in Portugal; further to characterize other associated resistance genes and their plasmid content, the phylogenetic groups, and the clonal relationship of ESBL-positive isolates. Sixty-five fecal samples were seeded in Levine media supplemented with cefotaxime for E. coli recovery. Susceptibility to 16 antimicrobial agents was performed by disk diffusion agar. ESBL-phenotypic detection was carried out by double-disk test; and the presence of the genes encoding TEM, OXA, SHV, and CTX-M type beta-lactamases was studied by polymerase chain reaction and sequencing. Other mechanisms of antimicrobial resistance and phylogenetic groups were also determined. Clonal relationship was performed by pulsed-field gel electrophoresis. ESBL-producing E. coli isolates were detected in 16 fecal samples, and one isolate per sample was studied. The CTX-M-1 type ESBL was detected in the 16 isolates. The gene encoding TEM-1 was identified to be associated with eight CTX-M-1-positive isolates. The tet(A) gene was found in 12 of 14 tetracycline-resistant isolates, and the aadA or strA-strB genes were found in the streptomycin-resistant isolates. Fourteen and two ESBL-containing isolates belonged to A and B1 phylogenetic groups, respectively. Clonal relationship of ESBL-containing isolates identified seven unrelated patterns. Swine represent an important reservoir of ESBL-containing E. coli isolates, especially of the CTX-M-1 type.

Extended-spectrum beta-lactamase-containing Escherichia coli isolates were detected in 32 of 119 fecal samples (26.9%) from birds of prey at Serra da Estrela, and these isolates contained the following beta-lactamases: CTX-M-1 (n = 13), CTX-M-1 plus TEM-1 (n = 14), CTX-M-1 plus TEM-20 (n = 1), SHV-5 (n = 1), SHV-5 plus TEM-1 (n = 2), and TEM-20 (n = 1).

We sought to determine the resistance phenotypes for erythromycin and clindamycin and the mechanisms implicated in 93 Streptococcus agalactiae isolates recovered from healthy pregnant women. Susceptibility testing for erythromycin, clindamycin, penicillin, cefotaxime, vancomycin, quinupristin-dalfopristin, choramphenicol, ofloxacin, and meropenen was carried out by disc-diffusion test, and the E-test was also applied for erythromycin and clindamycin. The constitutive MLS(B) resistance (cMLS(B)) and inducible MLS(B) resistance (iMLS(B)) phenotypes, respectively, as well as the M resistance phenotype were determined by the erythromycin-clindamycin double-disc test. The presence of ermA, ermB, ermC, msrA, and mef(A/E) macrolide resistance genes was studied by PCR. Resistance to erythromycin and clindamycin was found in 15% and 9.6% of the isolates, respectively. The resistance phenotypes detected among the 14 erythromycin-resistant isolates were as follows (number of isolates): cMLS(B) (9), iMLS(B) (3), and M (2). The MICs for erythromycin and clindamycin were as follows: cMLS(B) isolates (128-256 and >or=32 mg/L, respectively), iMLS(B) isolates (16-256 and 1 mg/L), and M isolates (2-8 and 1 mg/L). The following combination of genes were detected among isolates with cMLS(B) or iMLS(B) phenotypes: erm(B) (6 isolates), ermA + ermTR (3), ermA + ermB + ermTR (1), and none of these genes (2). The two isolates with M phenotype harbored the mef(A/E), and msrA gene was also found in one of them.