Carmen Tenorio

Bacteriocin Production in Vancomycin-Resistant and Vancomycin-Susceptible Enterococcus Isolates of Different Origins

ABSTRACT Bacteriocin production was determined for 218Enterococcus isolates (Enterococcus faecalis[93] and E. faecium[125]) obtained from different origins (human clinical samples [87], human fecal samples [78], sewage [28], and chicken samples [25]) and showing different vancomycin susceptibility patterns (vancomycin resistant, all of them vanA positive [56], and vancomycin susceptible [162]). All enterococcal isolates were randomly selected except for the vancomycin-resistant ones. A total of 33 isolates of eight different bacterial genera were used as indicators for bacteriocin production. Forty-seven percent of the analyzed enterococcal isolates were bacteriocin producers (80.6% of E. faecalis and 21.6% ofE. faecium isolates). The percentage of bacteriocin producers was higher among human clinical isolates (63.2%, 81.8% of vancomycin-resistant isolates and 60.5% of vancomycin-susceptible ones) than among isolates from the other origins (28 to 39.3%). Only one out of the 15 vancomycin-resistant isolates from human fecal samples was a bacteriocin producer, while 44.4% of fecal vancomycin-susceptible isolates were. The bacteriocin produced by the vanA-containing E. faecium strain RC714, named bacteriocin RC714, was further characterized. This bacteriocin activity was cotransferred together with thevanA genetic determinant to E. faecalis strain JH2-2. Bacteriocin RC714 was purified to homogeneity and its primary structure was determined by amino acid sequencing, showing an identity of 88% and a similarity of 92% with the previously described bacteriocin 31 from E. faecalis YI717. The presence of five different amino acids in bacteriocin RC714 suggest that this could be a new bacteriocin. The results obtained suggest that the epidemiology of vancomycin resistance may be influenced by different factors, including bacteriocin production.

High-level penicillin resistance and penicillin-gentamicin synergy in Enterococcus faecium.

Thirty-seven Enterococcus faecium strains with different levels of penicillin susceptibility were studied in time-kill experiments with a fixed concentration (5 micrograms/ml) of gentamicin combined with different penicillin concentrations (6 to 600 micrograms/ml). Synergy was defined as a relative decrease in counts of greater than 2 log10 CFU per milliliter after 24 h of incubation when the combination of the antibiotics was compared with its most active component alone. The minimal synergistic penicillin concentrations found were 6 micrograms/ml for 16 of 16 strains for which penicillin MICs were < or = 25 micrograms/ml, 20 to 100 micrograms/ml for 14 of 17 strains for which penicillin MICs were 50 to 200 micrograms/ml, and 200 to 500 micrograms/ml for 4 of 4 strains for which MICs penicillin were > 200 micrograms/ml. Penicillin-gentamicin synergy was observed even in high-level penicillin-resistant E. faecium strains at penicillin concentrations close to one-half the penicillin MIC. The possibility of treating infections caused by high-level penicillin-resistant E. faecium strains with penicillin-gentamicin combinations in particular cases may depend on the penicillin levels attainable in vivo.

Mutations in Ribosomal Protein L16 and in 23S rRNA in Enterococcus Strains for Which Evernimicin MICs Differ

ABSTRACT Mutations in ribosomal protein L16 and in 23S rRNA were investigated in 22 Enterococcus strains of different species and for which the MICs of evernimicin differ (MICs, 0.023 to 16 μg/ml). Amino acid changes (Arg56His, Ile52Thr, or Arg51His) in protein L16 were found in seven strains, and a nucleotide G2535A mutation in 23S rRNA was found in 1 strain among 13 for which the MICs are ≥1 μg/ml.

Antimicrobial resistance in faecal Escherichia coli isolates from farmed red deer and wild small mammals. Detection of a multiresistant E. coli producing extended-spectrum beta-lactamase.

Eighty-nine Escherichia coli isolates recovered from faeces of red deer and small mammals, cohabiting the same area, were analyzed to determine the prevalence and mechanisms of antimicrobial resistance and molecular typing. Antimicrobial resistance was detected in 6.7% of isolates, with resistances to tetracycline and quinolones being the most common. An E. coli strain carrying blaCTX-M-1 as well as other antibiotic resistant genes included in an unusual class 1 integron (Intl1-dfrA16-blaPSE-1-aadA2-cmlA1-aadA1-qacH-IS440-sul3-orf1-mef(B)Δ-IS26) was isolated from a deer. The blaCTX-M-1 gene was transferred by conjugation and transconjugants also acquired an IncN plasmid. This strain was typed as ST224, which seems to be well adapted to both clinical and environmental settings. The phylogenetic distribution of the 89 strains varied depending on the animal host. This work reveals low antimicrobial resistance levels among faecal E. coli from wild mammals, which reflects a lower selective pressure affecting these bacteria, compared to livestock. However, it is remarkable the detection of a multi-resistant ESBL-E. coli with an integron carrying clinically relevant antibiotic-resistance genes, which can contribute to the dissemination of resistance determinants among different ecosystems.

Ecology of Indigenous Lactic Acid Bacteria along Different Winemaking Processes of Tempranillo Red Wine from La Rioja (Spain)

Ecology of the lactic acid bacteria (LAB) during alcoholic fermentation (AF) and spontaneous malolactic fermentation (MLF) of Tempranillo wines from four wineries of La Rioja has been studied analyzing the influence of the winemaking method, processing conditions, and geographical origin. Five different LAB species were isolated during AF, while, during MLF, only Oenococcus oeni was detected. Although the clonal diversity of O. oeni strains was moderate, mixed populations were observed, becoming at least one strain with distinct PFGE profile the main responsible for MLF. Neither the winemaking method nor the cellar situation was correlated with the LAB diversity. However, processing conditions influenced the total number of isolates and the percentage of each isolated species and strains. The winemaking method could cause that genotypes found in semicarbonic maceration did not appear in other wineries. Four genotypes of O. oeni were isolated in more than one of the rest wineries. These four together with other dominant strains might be included in a future selection process.

Study of vancomycin resistance in faecal enterococci from healthy humans and dogs in Spain a decade after the avoparcin ban in Europe.

One hundred and 26 faecal samples from healthy dogs (2009) and 157 faecal samples from healthy humans (2007) from La Rioja region (Spain) were tested to know the carriage of vancomycin‐resistant enterococci (VRE). VRE with intrinsic resistance (vanC) were found in 12% of healthy dogs and humans (29 Enterococcus gallinarum and four Enterococcus casseliflavus). Nevertheless, VRE with acquired mechanism of resistance were not detected among these samples. Four Enterococcus faecalis isolates with vancomycin MIC of 8‐16 mg L−1 were recovered in human samples, but no single organism with known mechanism of acquired resistance could be identified. These 37 VRE isolates (33 E. gallinarum/E. casseliflavus and four E. faecalis) of dog and human origin were further characterized (PCR detection of antibiotic resistance, virulence and bacteriocin genes). High prevalence of tetracycline resistance was identified (70%), especially among dog isolates harbouring tet(M) ± tet(L) genes; erythromycin resistance was also higher among isolates from dogs and they harboured the erm(B) gene, associated with erm(A) gene in one case. Virulence genes were only identified among E. faecalis isolates of human origin (agg, cpd and/or gelE) and never among E. gallinarum/E. casseliflavus of human or dog origin. Five E. gallinarum isolates of dog and three E. faecalis of human origin expressed bacteriocin activity; among them, only one E. faecalis presented activity against Listeria monocytogenes. The bacteriocin structural gene ef1097 was identified in 3 bacteriocin‐producing E. faecalis isolates, associated with ent1071 in one of them.

Characterization of the Mechanisms of Fluoroquinolone Resistance in Vancomycin-Resistant Enterococci of Different Origins

Abstract The mutations in gyrA and parC genes were analyzed in 22 vancomycin-resistant enterococci of different origins and species, which had varying susceptibility to ciprofloxacin (minimum inhibitory concentration, MIC: 0.5->256 mg/L). All vanA or vanB2-containing strains with ciprofloxacin MIC of >32 mg/L presented amino acid changes in GyrA protein (S83I, S83Y, S83R or S83IE87G) with/without changes in parC protein (S80I or S80R or S80L). Strains with lower ciprofloxacin MICs presented the GyrA and parC wild type. One vanA-containing Enterococcus durans strain with a ciprofloxacin MIC of 64 mg/L presented the S83i and S80i changes in GyrA and parC proteins, respectively. Two vanB2 Enterococcus faecium strains were typed by multi-locus-sequencetyping and both were ascribed to the CC17 clonal complex with two sequence-types (ST78 and ST17-like). All seven vancomycin-resistant and ciprofloxacin-resistant E. faecium strains showed ampicillin resistance (MIC 32-256 mg/L), identifying the following amino acid changes in PBP5 protein: Q461K, V462K, H470Q, M485A, N496K, A499T, E525D, N546T, A558T, G582S, K632Q, P642l, E629V and P667S, together with a serine insertion at position 466′. The 12 Enterococcus gallinarum and Enterococcus casseliflavus isolates included in the study exhibited an MIC for ciprofloxacin in the range 0.5-16 mg/L and no amino acid changes were identified in GyrA or parC proteins. Specific mutations in gyrA and parC genes are associated with fluoroquinolone resistance in E. faecium and E. durans of different origins.

Potential spoilage yeasts in winery environments: Characterization and proteomic analysis of Trigonopsis cantarellii

Wine microbiota is complex and includes a wide diversity of yeast species. Few of them are able to survive under the restrictive conditions of dry red wines. In our study we detected and identified seven yeast species of the order Saccharomycetales that can be considered potential spoilers of wines due to physiological traits such as acidogenic metabolism and off-odor generation: Arthroascus schoenii, Candida ishiwadae, Meyerozyma guilliermondii, Pichia holstii, Pichia manshurica, Trigonopsis cantarellii, and Trigonopsis variabilis. Based on the prevalence of T. cantarellii isolates in the wine samples of our study, we further characterized this species, determined molecular and phenotypic features, and performed a proteomic analysis to identify differentially expressed proteins at mid-exponential growth phase in the presence of ethanol in the culture broth. This yeast species is shown to be able to grow in the presence of ethanol by expressing heat shock proteins (Hsp70, Hsp71) and a DNA damage-related protein (Rad24), and to be able to confer spoilage characteristics on wine.

Transcriptome analysis shows activation of the arginine deiminase pathway in Lactococcus lactis as a response to ethanol stress

This paper describes the molecular response of Lactococcus lactis NZ9700 to ethanol. This strain is a well-known nisin producer and a lactic acid bacteria (LAB) model strain. Global transcriptome profiling using DNA microarrays demonstrated a bacterial adaptive response to the presence of 2% ethanol in the culture broth and differential expression of 67 genes. The highest up-regulation was detected for those genes involved in arginine degradation through the arginine deiminase (ADI) pathway (20-40 fold up-regulation). The metabolic responses to ethanol of wild type L. lactis strains were studied and compared to those of regulator-deletion mutants MG∆argR and MG∆ahrC. The results showed that in the presence of 2% ethanol those strains with an active ADI pathway reached higher growth rates when arginine was available in the culture broth than in absence of arginine. In a chemically defined medium strains with an active ADI pathway consumed arginine and produced ornithine in the presence of 2% ethanol, hence corroborating that arginine catabolism is involved in the bacterial response to ethanol. This is the first study of the L. lactis response to ethanol stress to demonstrate the relevance of arginine catabolism for bacterial adaptation and survival in an ethanol containing medium.

Detection of aminoglycoside-penicillin synergy against Enterococcus faecium using high-content aminoglycoside disks

Thirty-sevenEnterococcus faecium strains were screened for high-level aminoglycoside resistance with an agar diffusion test using high-content aminoglycoside disks (300 μg of streptomycin and 120 μg of gentamicin, tobramycin, kanamycin or amikacin). The inhibition zones obtained were correlated with results of time-kill penicillin-aminoglycoside synergy studies. An 11 mm breakpoint differentiated strains susceptible or resistant to the synergy of streptomycin plus penicillin. Irrespective of the inhibition zones obtained with tobramycin and kanamycin disks,Enterococcus faecium strains never showed synergy with penicillin in combination with these aminoglycosides. Penicillin-amikacin synergy cannot be predicted by the amikacin disks. Nevertheless, even though kanamycin disks do not predict penicillin-kanamycin synergy, they can be used to predict penicillinamikacin synergy. In summary, high-content streptomycin, gentamicin and kanamycin disks can be used to predict the susceptibility ofEnterococcus faecium strains to the synergistic combination of penicillin plus one of the aminoglycosides (streptomycin, gentamicin or amikacin, respectively).