Antibiotic resistance and virulence of faecal enterococci isolated from food-producing animals in Tunisia

Antimicrobial agents exert a selection pressure not only on pathogenic, but also on commensal bacteria of the intestinal tract of humans and animals. The aim of this work was to determine the occurrence of different enterococcal species and to analyse the prevalence of antimicrobial resistance and the mechanisms implicated, as well as the genetic diversity in enterococci recovered from faecal samples of food-producing animals (poultry, beef and sheep) in Tunisia. Antimicrobial resistance and the mechanisms implicated were studied in 87 enterococci recovered from 96 faecal samples from animals of Tunisian farms. Enterococcus faecium was the most prevalent species detected (46 %), followed by E. hirae (33.5 %). High percentages of resistance to erythromycin and tetracycline were found among our isolates, and lower percentages to aminoglycosides and ciprofloxacin were identified. Most of the tetracycline-resistant isolates carried the tet(M) and/or tet(L) genes. The erm(B) gene was detected in all erythromycin-resistant isolates. The ant(6)-Ia, aph(3′)-Ia and aac(6′)-aph(2″) genes were detected in nine aminoglycoside-resistant isolates. Of our isolates, 11.5 % carried the gelE gene and exhibited gelatinase acitivity. The esp gene was detected in 10 % of our isolates and the hyl gene was not present in any isolate. The predominant species (E. faecium and E. hirae) showed a high genetic diversity by repetitive extragenic palindromic (REP)-PCR. Food animals might play a role in the spread through the food chain of enterococci with virulence and resistance traits to humans.     

Antibiotic resistance and virulence factors among clinical and food enterococci isolated in Slovakia

The resistance to antibiotics and the distribution of virulence factors in enterococci isolated from traditional Slovak sheep cheese bryndza was compared with strains from human infections. The occurrence of 4 enterococcal species was observed in 117 bryndza-cheese isolates. The majority of strains were identified as E. faecium (76 %) and E. faecalis (23 %). Several strains of E. durans and 1 strain of E. hirae were also present. More than 90 % of strains isolated from 109 clinical enterococci were E. faecalis, the rest belonged to E. faecium. The resistance to 6 antimicrobial substances (ampicillin, ciprofloxacin, higher concentration of gentamicin, nitrofurantoin, tetracycline and vancomycin) was tested in clinical and food enterococci. A higher level of resistance was found in clinical than in food strains and E. faecium had a higher resistance than E. faecalis; no resistance to vancomycin was detected. The occurrence of 3 virulence-associated genes, cylA (coding for hemolysin), gelE (coding for gelatinase) and esp (coding for surface protein) was monitored. Differences were found in the distribution of cylA gene between clinical and bryndza-cheese E. faecalis strains; in contrast to clinical strains (45 %), cylA gene was detected in 22 % of food isolates. The distribution of 2 other virulence factors, gelE and esp, was not significantly different in the two groups of E. faecalis strains. cylA and gelE genes were not detected in E. faecium but more than 70 % of clinical E. faecium were positive for esp, even thought none of the 79 E. faecium cheese isolates contained this gene.     

Antibiotic resistance and genotypic characterization by PFGE of clinical and environmental isolates of enterococci

Fifty-four Enterococcus faecalis and 20 Enterococcus faecium isolates from clinical and non-human sources in Rome, Italy, were characterized by antibiotic resistance and pulsed field gel electrophoresis (PFGE). Resistance to vancomycin, teicoplanin, ampicillin, and ciprofloxacin was more frequent in E. faecium than in E. faecalis, whereas high-level resistance to aminoglycoside was found primarily in E. faecalis. Multi-resistance was found primarily among clinical isolates, but was also observed among environmental isolates. Common genotypes shared among clinical and environmental isolates were observed, however, the majority of isolates occurred as unique, source-specific clones. Several PFGE types were associated with shared features in their antibiotic resistance patterns; evidences of clonal spread between and within wards were also noted. This is the first report indicating clonal relatedness between human and environmental enterococci isolated in Italy.     

Diversity of species and antibiotic resistance in enterococci isolated from seafood in Tunisia

The purpose of this study was to analyse the antibiotic resistance and virulence of enterococci recovered from seafood and to characterise the associated genes. Forty-four enterococcal isolates [Enterococcus faecalis (21), E. faecium (11), E. casseliflavus (5), E. durans (3), E. hirae (2), E. gallinarum (1) and E. mundtii (1)] were recovered from 70 samples of seafood collected during March–May 2015 in Tunisia. Isolates were tested for antibiotic resistance to 12 antibiotics by the disc diffusion method. Rates of resistance in the range 25–45.5% were observed for pristinamycin, ciprofloxacin, streptomycin, tetracycline and erythromycin, and in the range 6.8–9.1% for kanamycin, gentamicin and chloramphenicol. However, all strains showed susceptibility to β-lactams and glycopeptides. Multi-resistance to at least three different classes of antibiotics was detected in 14 strains (31.8%). Among 12 tetracycline-resistant enterococci, tet(M) was detected in 11 isolates and tet(L) in seven isolates. The erm(B) gene was identified in 91% of erythromycin-resistant isolates. All chloramphenicol-resistant isolates carried the cat gene, and all kanamycin-resistant isolates harboured the aph(3)-IIIa gene. The aac(6′)-aph(2″) and ant(6)-Ia genes were detected in high-level gentamicin- and streptomycin-resistant isolates, respectively. The virulence genes gelE (29.5%), esp (9.1%), cylA and cylB (9.1%) were found in enterococci. This is the first study in Tunisia to underscore the importance of seafood as a reservoir of enterococci carrying resistance and virulence genes.     

Antibiotic resistance and virulence traits in clinical and environmental Enterococcus faecalis and Enterococcus faecium isolates

This study compared virulence and antibiotic resistance traits in clinical and environmental Enterococcus faecalis and Enterococcus faecium isolates. E. faecalis isolates harboured a broader spectrum of virulence determinants compared to E. faecium isolates. The virulence traits Cyl-A, Cyl-B, Cyl-M, gel-E, esp and acm were tested and environmental isolates predominantly harboured gel-E (80% of E. faecalis and 31.9% of E. faecium) whereas esp was more prevalent in clinical isolates (67.8% of E. faecalis and 70.4% of E. faecium). E. faecalis and E. faecium isolated from water had different antibiotic resistance patterns compared to those isolated from clinical samples. Linezolid resistance was not observed in any isolates tested and vancomycin resistance was observed only in clinical isolates. Resistance to other antibiotics (tetracycline, gentamicin, ciprofloxacin and ampicillin) was detected in both clinical and water isolates. Clinical isolates were more resistant to all the antibiotics tested compared to water isolates. Multi-drug resistance was more prevalent in clinical isolates (71.2% of E. faecalis and 70.3% of E. faecium) compared to water isolates (only 5.7% E. faecium). tet L and tet M genes were predominantly identified in tetracycline-resistant isolates. All water and clinical isolates resistant to ciprofloxacin and ampicillin contained mutations in the gyrA, parC and pbp5 genes. A significant correlation was found between the presence of virulence determinants and antibiotic resistance in all the isolates tested in this study (p<0.05). The presence of antibiotic resistant enterococci, together with associated virulence traits, in surface recreational water could be a public health risk.     

Antimicrobial resistance in faecal enterococci and Escherichia coli isolates recovered from Iberian wolf

The aim of this study was to report the antimicrobial resistance, the molecular mechanisms associated and the detection of virulence determinants within faecal Enterococcus spp. and Escherichia coli isolates of Iberian wolf. Enterococci (n = 227) and E. coli (n = 195) isolates were obtained from faecal samples of Iberian wolf (Canis lupus signatus). High rates of resistance were detected for tetracycline and erythromycin among the enterococci isolates, and most of resistant isolates harboured the tet(M) and/or tet(L) and erm(B) genes, respectively. The bla_TEM, tet(A) and/or tet(B), and aadA or strA‐strB genes were detected among most ampicillin‐, tetracycline‐ or streptomycin‐resistant E. coli isolates, respectively. E. coli isolates were ascribed to phylogroups A (n = 56), B1 (91), B2 (13) and D (35). The occurrence of resistant enterococci and E. coli isolates in the faecal flora of Iberian wolf, including the presence of resistant genes in integrons, and virulence determinants was showed in this study. Iberian wolf might act as reservoir of certain resistance genes that could be spread throughout the environment.

Significance and Impact of the Study

This study shows antimicrobial resistance in commensal bacteria from the free‐range, Portuguese, Iberian wolf population. The results indicate that the Iberian wolf could contribute to the spread of resistant bacteria throughout the environment. Additionally, in case of infection, an increased risk of therapeutic failure due to the presence of multiresistant bacteria may represent a health problem for this endangered species. Future studies must be performed to analyse the possible contamination of these animals through the environment and/or the food chain.     

Streptogramin resistance patterns and virulence determinants in vancomycin-susceptible enterococci isolated from multi-component deli salads

Aims: This study examined vancomycin-susceptible Enterococcus (VSE) from deli salads for streptogramin resistance and presence of cpd, agg and gelE genes. Methods and Results: Fifteen VSE from retail salads were isolated for identification and antimicrobial susceptibility testing by MicroScan, Etest and agar diffusion. Clinical vancomycin-resistant Enterococcus (n = 32) and animal VSE (n = 17) were included for comparative purposes. Multiplex PCR was used to detect the following genes: agg, gelE, cpd, vatD, vatE and sodA. Results showed fewer streptogramin-susceptible Enterococcus faecium isolated from salad (1/6, 17%) and animals (6/10, 60%) than from clinical (26/29, 90%) sources. A low level of erythromycin susceptibility was detected among salad (2/6, 33%) and animal (3/10, 30%) Ent. faecium isolates. Food and animal VSE demonstrated similarities in antimicrobial resistance profiles. All Enterococcus faecalis carried one or more of the selected genes cpd (40%), gelE (33%) and agg (27%). The vatD or vatE genes were not detected in any of the isolates. Conclusions: Experiments demonstrated that streptogramin resistance and virulence genes agg, cpd and gelE are present in enterococci isolated from deli salads. Significance and Impact of the Study: This study provides useful information regarding streptogramin resistance and virulence determinants in enterococci from foods associated with multi-component ingredients.     

Chemiluminescence of enterococci isolates from freshwater

All Enterococcus spp., isolated from environmental water samples (n=81), emitted a high chemiluminescence signal in the presence of luminol (10(-2) M). Kinetic studies of chemiluminescence show a close correlation between chemiluminescence and growth curves during the exponential phase, with a maximum chemiluminescence reached just before bacterial growth entered in the stationary phase. On the other hand, genera closely related to Enterococcus such as Streptococcus or Lactococcus produced a very weak chemiluminescent signal. Chemiluminescence of enterococci could therefore offer a rapid test, in aiding the identification of the genus Enterococcus and in the survey of the microbiological quality of water supplies.     

Antibiotic use in humans and bacterial resistance.

As with any public health problem, the evolution of antibacterial resistance must be viewed from a perspective of risk, and analysed in terms of probabilities within the populations. It is necessary to be able to predict the risk of antibacterial resistance, in the future, and two main strategies have recently been developed in mathematical models that may help to evaluate these risks. It is also important to understand how antibiotics are used and how their use affects the evolution of antibacterial resistance. Understanding the epidemiology of antibacterial resistance will enable us to develop preventive strategies to limit existing resistance and to avoid the emergence of new strains of resistant bacteria.     

Antimicrobial resistance and virulence genes of Escherichia coli isolates from swine in Ontario

A total of 318 Escherichia coli isolates obtained from diarrheic and healthy pigs in Ontario from 2001 to 2003 were examined for their susceptibility to 19 antimicrobial agents. They were tested by PCR for the presence of resistance genes for tetracycline, streptomycin, sulfonamides, and apramycin and of 12 common virulence genes of porcine E. coli. Antimicrobial resistance frequency among E. coli isolates from swine in Ontario was moderate in comparison with other countries and was higher in isolates from pigs with diarrhea than in isolates from healthy finisher pigs. Resistance profiles suggest that cephamycinases may be produced by > or = 8% of enterotoxigenic E. coli (ETEC). Resistance to quinolones was detected only in enterotoxigenic E. coli (< or = 3%). The presence of sul3 was demonstrated for the first time in Canada in porcine E. coli isolates. Associations were observed among tetA, sul1, aadA, and aac(3)IV and among tetB, sul2, and strA/strB, with a strong negative association between tetA and tetB. The paa and sepA genes were detected in 92% of porcine ETEC, and strong statistical associations due to colocation on a large plasmid were observed between tetA, estA, paa, and sepA. Due at least in part to gene linkages, the distribution of resistance genes was very different between ETEC isolates and other porcine E. coli isolates. This demonstrates that antimicrobial resistance epidemiology differs significantly between pathogenic and commensal E. coli isolates. These results may have important implications with regards to the spread and persistence of resistance and virulence genes in bacterial populations and to the prudent use of antimicrobial agents.     

Antibiotic resistance dynamics and isolation rate of staphylococci and enterococci from patients of reconstructive surgery units

The dynamics of isolation of staphylococci and enterococci from clinical material of patients and their antibiotic susceptibility within a 5-year period (2005-2009) was analysed. 5990 isolates were tested: 1250 isolates of Staphylococcus aureus, 3268 isolates of S. epidermidis, 1005 isolates of Enterococcus faecalis and 467 isolates of E. faecium. Grampositive infections were shown to be prevailing within the last 2-3 years, the nosocomial epidermal staphylococci more and more replacing S. aureus (the ratio of S. epidermidis and S. aureus in 2009 was 3.3). The isolation rate of E. faecalis significantly increased (by 3.5 times) and the ratio of E. faecalis and E. faecium in 2009 was 4.3. The microflora composition with respect to the isolation source was analysed and its clinical significance was estimated. The study of the antibiotic susceptibility showed that oxacillin had its own specific niche, while antibiotics active against resistant grampositive cocci, such as rifampicin, fusidic acid, fluoroquinolones (moxifloxacin), cefoxitin, as well as amoxicillin/clavulane in infections due to E. faecalis, might be considered as the drugs of choice. In the treatment of nosocomial infections, when the etiological role of MRSA or VRE is suspected or confirmed, the complex therapy should obligatory include the most active antibiotics (vancomycin or linezolid among them).     

Antimicrobial resistance of Escherichia coli isolates from broiler chickens and humans

Background

Inguinal hernias are usually caused by a congenital defect, which occurs as a weakness of the inguinal canal. Porcine β-glucuronidase gene (GUSB) was chosen as functional candidate gene because of its involvement in degradation of hyaluronan within gubernacular tissue during descent of testes. Since a genome-wide linkage analysis approach has shown evidence that two regions on porcine chromosome 3 (SSC 3) are involved in the inheritance of hernia inguinalis/scrotalis in German pig breeds, GUSB also attained status as a positional candidate gene by its localization within a hernia-associated chromosomal region.

Results

A contig spanning 17,157 bp, which contains the entire GUSB, was assembled. Comparative sequence analyses were conducted for the GUSB gene locus. Single nucleotide polymorphisms (SNPs) located within the coding region of GUSB were genotyped in 512 animals. Results of transmission disequilibrium test (TDT) for two out of a total of five detected SNPs gave no significant association with the outcome of hernia in pigs.

Conclusion

On the basis of our studies we are able to exclude the two analyzed SNPs within the porcine GUSB gene as causative for the transmission of inguinal hernia.     

Occurrence of high-level aminoglycoside resistance in environmental isolates of enterococci.

High-level resistance to aminoglycosides was observed in environmental isolates of enterococci. Various aquatic habitats, including agricultural runoff, creeks, rivers, wastewater, and wells, were analyzed. Strains of Enterococcus faecalis, E. faecium, E. gallinarum, and other Enterococcus spp. demonstrated multiple antibiotic resistance patterns to aminoglycosides.     

Antibiotic resistance of enterococci and related bacteria in an urban wastewater treatment plant

The main objective of this work was to study the ecology of enterococci and related bacteria in raw and treated wastewater from a treatment plant receiving domestic and pretreated industrial effluents in order to assess the influence of treatment on the prevalence of antibiotic resistance phenotypes among this group of bacteria. The predominant species found in the raw wastewater were Entercoccus hirae, Entercoccus faecium and Entercoccus faecalis. Wastewater treatment led to a reduction in E. hirae (alpha<0.1) and an increase in E. faecium (alpha<0.1); the relative proportions of E. faecalis remained the same in the raw and in the treated wastewater. Among the isolates tested, no vancomycin resistance was observed among the enterococci. Entercoccus faecium and E. faecalis showed resistance prevalence values reaching 33%, 40% and 57% for the antibiotics ciprofloxacin, erythromycin and tetracycline, respectively. Antibiotic-resistant strains of enterococci were not eliminated by wastewater treatment. A positive selection of ciprofloxacin-resistant enterococci was indicated by a significant increase in resistance prevalence (alpha<0.02) in treated wastewater compared with the raw wastewater.