Detection of linezolid and vancomycin resistant Enterococcus isolates collected from healthy chicken caecum.

AIM: The poultry industry represents an important economic sector in Tunisia. This study aims to determine the antimicrobial resistance phenotypes and genotypes and virulence factors of enterococci collected from chicken caecum in Tunisia. METHODS AND RESULTS: Forty-nine composite chicken caecum samples were recovered in 49 different Tunisian farms (December 2019-March 2020). Each composite sample corresponds to six individual caecum from each farm. Composite samples were plated on Slanetz-Bartley agar both supplemented (SB-Van) and not supplemented (SB) with vancomycin and isolates were identified by matrix-assisted laser desorption/ionization time-of-flight. Antibiotic resistance and virulence genes were tested by Polymerase Chain Reaction (PCR) and sequencing and multilocus-sequence-typing of selected enterococci was performed. One hundred sixty seven enterococci of six different species were recovered. Acquired linezolid resistance was detected in 6 enterococci of 4/49 samples (8.1%): (A) four optrA-carrying Enterococcus faecalis isolates assigned to ST792, ST478, and ST968 lineages; (B) two poxtA-carrying Enterococcus faecium assigned to ST2315 and new ST2330. Plasmid typing highlighted the presence of the rep10, rep14, rep7, rep8, and pLG1 in these strains. One vancomycin-resistant E. faecium isolate (typed as ST1091) with vanA gene (included in Tn1546) was detected in SB-Van plates. The gelE, agg, esp, and hyl virulence genes were found in linezolid- and vancomycin-resistant enterococci. High resistance rates were identified in the enterococci recovered in SB plates: tetracycline [74.8%, tet(M) and tet(L) genes], erythromycin [65.9%, erm(B)], and gentamicin [37.1%, aac(6')-Ie-aph(2″)-Ia]. CONCLUSION: The detection of emerging mechanisms of resistance related to linezolid and vancomycin in the fecal enterococci of poultry farms has public health implications, and further surveillance should be carried out to control their dissemination by the food chain.

Antimicrobial Activity of Synthetic Enterocins A, B, P, SEK4, and L50, Alone and in Combinations, against Clostridium perfringens.

Multidrug-resistant Clostridium perfringens infections are a major threat to the poultry industry. Effective alternatives to antibiotics are urgently needed to prevent these infections and limit the spread of multidrug-resistant bacteria. The aim of the study was to produce by chemical synthesis a set of enterocins of different subgroups of class II bacteriocins and to compare their spectrum of inhibitory activity, either alone or in combination, against a panel of twenty C. perfringens isolates. Enterocins A, P, SEK4 (class IIa bacteriocins), B (unsubgrouped class II bacteriocin), and L50 (class IId leaderless bacteriocin) were produced by microwave-assisted solid-phase peptide synthesis. Their antimicrobial activity was determined by agar well diffusion and microtitration methods against twenty C. perfringens isolates and against other pathogens. The FICINDEX of different combinations of the selected enterocins was calculated in order to identify combinations with synergistic effects. The results showed that synthetic analogs of L50A and L50B were the most active against C. perfringens. These peptides also showed the broadest spectrum of activity when tested against other non-clostridial indicator strains, including Listeria monocytogenes, methicillin-resistant Staphylococcus aureus, Streptococcus suis, Streptococcus pyogenes, Enterococcus cecorum, Enterococcus faecalis, as well as Gram-negative bacteria (Campylobacter coli and Pseudomonas aeruginosa), among others. The selected synthetic enterocins were combined on the basis of their different mechanisms of action, and all combinations tested showed synergy or partial synergy against C. perfringens. In conclusion, because of their high activity against C. perfringens and other pathogens, the use of synthetic enterocins alone or as a consortium can be a good alternative to the use of antibiotics in the poultry sector.

Pathogenicity and Antibiotic Resistance Diversity in Clostridium perfringens Isolates from Poultry Affected by Necrotic Enteritis in Canada.

Necrotic enteritis (NE) caused by C. perfringens is one of the most common diseases of poultry and results in a huge economic loss to the poultry industry, with resistant clostridial strains being a serious concern and making the treatment difficult. Whole-genome sequencing approaches represent a good tool to determine resistance profiles and also shed light for a better understanding of the pathogen. The aim of this study was to characterize, at the genomic level, a collection of 20 C. perfringens isolates from poultry affected by NE, giving special emphasis to resistance mechanisms and production of bacteriocins. Antimicrobial resistance genes were found, with the tet genes (associated with tetracycline resistance) being the most prevalent. Interestingly, two isolates carried the erm(T) gene associated with erythromycin resistance, which has only been reported in other Gram-positive bacteria. Twelve of the isolates were toxinotyped as type A and seven as type G. Other virulence factors encoding hyaluronases and sialidases were frequently detected, as well as different plasmids. Sequence types (ST) revealed a high variability of the isolates, finding new allelic combinations. Among the isolates, C. perfringens MLG7307 showed unique characteristics; it presented a toxin combination that made it impossible to toxinotype, and, despite being identified as C. perfringens, it lacked the housekeeping gene colA. Genes encoding bacteriocin BCN5 were found in five isolates even though no antimicrobial activity could be detected in those isolates. The bcn5 gene of three of our isolates was similar to one previously reported, showing two polymorphisms. Concluding, this study provides insights into the genomic characteristics of C. perfringens and a better understanding of this avian pathogen.

Detection of antimicrobial producing Staphylococcus from migratory birds: Potential role in nasotracheal microbiota modulation.

A collection of 259 staphylococci of 13 different species [212 coagulase-negative (CoNS) and 47 coagulase-positive (CoPS)] recovered from nasotracheal samples of 87 healthy nestling white storks was tested by the spot-on-lawn method for antimicrobial-activity (AA) against 14 indicator bacteria. Moreover, extracts of AP isolates were obtained [cell-free-supernatants (CFS) both crude and concentrated and butanol extracts] and tested against the 14 indicator bacteria. The microbiota modulation capacity of AP isolates was tested considering: (a) intra-sample AA, against all Gram-positive bacteria recovered in the same stork nasotracheal sample; (b) inter-sample AA against a selection of representative Gram-positive bacteria of the nasotracheal microbiota of all the storks (30 isolates of 29 different species and nine genera). In addition, enzymatic susceptibility test was carried out in selected AP isolates and bacteriocin encoding genes was studied by PCR/sequencing. In this respect, nine isolates (3.5%; seven CoNS and two CoPS) showed AA against at least one indicator bacteria and were considered antimicrobial-producing (AP) isolates. The AP isolates showed AA only for Gram-positive bacteria. Three of these AP isolates (S. hominis X3764, S. sciuri X4000, and S. chromogenes X4620) revealed AA on all extract conditions; other four AP isolates only showed activity in extracts after concentration; the remaining two AP isolates did not show AA in any of extract conditions. As for the microbiota modulation evaluation, three of the nine AP-isolates revealed intra-sample AA. It is to highlight the potent inter-sample AA of the X3764 isolate inhibiting 73% of the 29 representative Gram-positive species of the nasotracheal stork microbiota population. On the other hand, enzymatic analysis carried out in the two highest AP isolates (X3764 and X4000) verified the proteinaceous nature of the antimicrobial compound and PCR analysis revealed the presence of lantibiotic-like encoding genes in the nine AP isolates. In conclusion, these results show that nasotracheal staphylococci of healthy storks, and especially CoNS, produce antimicrobial substances that could be important in the modulations of their nasal microbiota.

Targeting Enterococci with Antimicrobial Activity against Clostridium perfringens from Poultry.

Necrotic enteritis (NE), caused by Clostridium perfringens, is an emerging issue in poultry farming. New approaches, other than antibiotics, are necessary to prevent NE development and the emergence of multidrug-resistant bacteria. Enterococci are commensal microorganisms that can produce enterocins, antimicrobial peptides with activities against pathogens, and could be excellent candidates for protective cultures. This study aimed to screen and characterize Enterococcus strains of poultry origin for their inhibitory activity against C. perfringens. In total, 251 Enterococcus strains of poultry origin plus five bacteriocin-producing (BP+) E. durans strains of other origins were screened for antimicrobial activity against the indicator C. perfringens X2967 strain using the "spot on the lawn" method. We detected thirty-two BP+ strains (eleven Enterococcus faecium, nine E. gallinarum, eight E. faecalis, three E. durans, and one E. casseliflavus). We further studied the antimicrobial activity of the supernatants of these 32 BP+ strains using agar well diffusion and microtitration against a collection of 20 C. perfringens strains. Twelve BP+ enterococci that were found to exhibit antimicrobial activity against C. perfringens were characterized using whole genome sequencing. Among these, E. faecium X2893 and X2906 were the most promising candidates for further studies as protective cultures for poultry farming. Both strains belong to the sequence type ST722, harbor the genes encoding for enterocin A and enterocin B, do not possess acquired resistance genes, do not carry plasmids, and present the acm gene, which is implicated in host colonization. Further research is needed to determine the utility of these strains as protective cultures.

Airborne Dissemination of Bacteria (Enterococci, Staphylococci and Enterobacteriaceae) in a Modern Broiler Farm and Its Environment.

The role of the air as a vehicle of bacteria dissemination in the farming environment has been previously reported, but still scarcely studied. This study investigated the bacteria density/diversity of the inside and outside air and of litter samples at a broiler farm. Samples were collected considering two seasons, three outside air distances (50/100/150 m) and the four cardinal directions. Selective media was used for staphylococci, enterococci, and Enterobacteriaceae recovery. A high number of bacteria was detected in the litter (2.9 × 105-5.8 × 107 cfu/g) and in the inside air (>105 cfu/m3), but a low emission of bacteria was evidenced in the outside air (<6 cfu/m3). Moreover, the bacteria detected in the farm's outside air decreased the further from the farm the sample was taken. A total of 544 isolates were identified by MALDI-TOF (146 from the litter, 142 from inside air and 256 from outside air). From these, 162 staphylococci (14 species; S. saprophyticus 40.7%), 176 Enterobacteriaceae (4 species; E. coli 66%) and 190 enterococci (4 species; E. hirae 83%) were detected. E. hirae was the predominant species, and identical PFGE clones were detected in inside and outside samples. The detection of identical DNA profiles in E. hirae isolates from inside and outside samples suggests the role of the air in bacterial dissemination from the inside of the broiler farm to the immediate environment.