Species distribution and genes encoding antimicrobial resistance in enterococcus spp. isolates from rabbits residing in diverse ecosystems: A new reservoir of linezolid and vancomycin resistance.

AIMS: Worldwide, studies regarding antimicrobial resistance in rabbits are scarce. In addition, it seems that rearing conditions have important impact on emergence and spread of antimicrobial-resistant bacteria. Thus, the authors sought to (1) assess the role of rabbits residing across diverse ecosystems as potential reservoirs of antimicrobial-resistant enterococci and (2) investigate the genetic background of detected resistances. METHODS AND RESULTS: Faecal samples from 60 healthy farmed rabbits (one farm), 35 laboratory rabbits and 31 wild rabbits were analysed. Overall, 97 enterococci isolates were accumulated, as follows: 44 E. faecium, 37 E. faecalis, 7 E. gallinarum, 5 E. durans and 4 E. avium. E. faecalis isolates were statistically associated with farm rabbits and wild rabbits (p < 0.05). High rates of resistance were observed for tetracycline (60.8%; tetM [n = 48; 81.3%], tetO [n = 7; 11.8%] and tetL [n = 1; 1.7%]), erythromycin (43.3%; msr(A) [n = 14; 33.3%] and ermB [n = 13; 31%]), ampicillin (29.9%), streptomycin (26.8%; ant(6)-Ia [n = 3, 11.5%]) and vancomycin (21.6%; vanA [one E. faecium + one E. faecalis; 9.5%]). Low frequencies of resistance were observed for teicoplanin (9.2%), linezolid (8.2%), ciprofloxacin (7.2%) and gentamicin (1%; aac(6')-Ie-aph(2″)-Ia). Resistance to ampicillin and vancomycin was associated with laboratory rabbits (p < 0.05). Int-Tn (Tn916/1545) was detected in 27 (27.8%) isolates, of which 10 isolates co-harboured tetM and ermB genes, while 16 comprised tetM. CONCLUSION: Findings indicate that clinically relevant enterococci species isolated from rabbits are frequently resistant to antimicrobials and harbour a range of genes associated with the Tn916/1545 family. SIGNIFICANCE AND IMPACT OF THE STUDY: This study highlights the high rates of antimicrobial-resistant enterococci from rabbits and the occurrence of both vancomycin- and linezolid-resistant isolates, potentially representing a very serious threat to human and animal health.

An Original and Efficient Antibiotic Adjuvant Strategy to Enhance the Activity of Macrolide Antibiotics against Gram-Negative Resistant Strains.

Gram-negative bacteria were reported as a significant cause of infections in both community and nosocomial settings. Considered as one of the greatest threats to public health, the spread of bacteria drug resistance and the lack of effective alternative treatment options remains problematic. Herein, we report a promising strategy to combat Gram-negative resistant strains consisting of the combination of a macrolide antibiotic with a polyaminoisoprenyl adjuvant derivative leading to a significant decrease of antibiotic resistance.

Valorization of Winemaking By-Products as a Novel Source of Antibacterial Properties: New Strategies to Fight Antibiotic Resistance.

The emergence of antibiotic-resistance in bacteria has limited the ability to treat bacterial infections, besides increasing their morbidity and mortality at the global scale. The need for alternative solutions to deal with this problem is urgent and has brought about a renewed interest in natural products as sources of potential antimicrobials. The wine industry is responsible for the production of vast amounts of waste and by-products, with associated environmental problems. These residues are rich in bioactive secondary metabolites, especially phenolic compounds. Some phenolics are bacteriostatic/bactericidal against several pathogenic bacteria and may have a synergistic action towards antibiotics, mitigating or reverting bacterial resistance to these drugs. Complex phenolic mixtures, such as those present in winemaking residues (pomace, skins, stalks, leaves, and especially seeds), are even more effective as antimicrobials and could be used in combined therapy, thereby contributing to management of the antibiotic resistance crisis. This review focuses on the potentialities of winemaking by-products, their extracts, and constituents as chemotherapeutic antibacterial agents.

Antimicrobial resistance genes and virulence gene encoding intimin in Escherichia coli and Enterococcus isolated from wild rabbits (Oryctolagus cuniculus) in Tunisia.

The spread of antimicrobial-resistant bacteria in wildlife must be viewed as a major concern with serious implications for human and animal health. Escherichia coli and enterococcal isolates were recovered from faecal samples of 49 wild rabbits (Oryctolagus cuniculus) on specific media and were characterised using biochemical and molecular tests. For all isolates, antimicrobial susceptibility testing was performed, and resistance genes were detected by PCR. Molecular typing of isolates was carried out by pulsed-field gel-electrophoresis, and E. coli strains were also tested for the presence of intimin (eae) gene characteristic of rabbit enteropathogenic E. coli. A total of 34 E. coli and 36 enterococci [E. hirae (52.8%) and E. faecalis (47.2%)] were obtained. For E. coli, resistance to tetracycline (94%), streptomycin (62%), ciprofloxacin (47%), trimethoprim-sulphamethoxazole (35%) and chloramphenicol (6%) was observed. Resistance to third-generation cephalosporins was detected in one E. coli strain that carried the blaCMY-2 and blaTEM-1 genes. Class 1 integrons were detected in eight isolates. For enterococci, resistance to tetracycline (63.9%), erythromycin (30.5%), streptomycin (18.2%), and chloramphenicol (5.5%) was detected. The tet(M)+tet(L), erm(B) and ant (6)-Ia genes were identified in thirteen, seven and three resistant Enterococcus strains, respectively. Molecular typing showed a high diversity among our strains. Wild rabbits could represent a reservoir of E. coli, and enterococci carrying antimicrobial resistance genes and E. coli additionally carrying the eae gene of enteropathogenic pathotypes could both contaminate the environment. our finding seems to represent the first report of eae-positive E. coli in wild rabbits.

Detection of CTX-M-15 harboring Escherichia coli isolated from wild birds in Tunisia.

BACKGROUND: The spreading of antibiotic resistant bacteria is becoming nowadays an alarming threat to human and animal health. There is increasing evidence showing that wild birds could significantly contribute to the transmission and spreading of drug-resistant bacteria. However, data for antimicrobial resistance in wild birds remain scarce, especially throughout Africa. The aims of this investigation were to analyze the prevalence of ESBL-producing E. coli in faecal samples of wild birds in Tunisia and to characterize the recovered isolates. RESULTS: One hundred and eleven samples were inoculated on MacConkey agar plates supplemented with cefotaxime (2 µg/ml). ESBL-producing E. coli isolates were detected in 12 of 111 faecal samples (10.81%) and one isolate per sample was further characterized. ß-lactamase detected genes were as follows: blaCTX-M-15 (8 isolates), blaCTX-M-15 + blaTEM-1b (4 isolates). The ISEcp1 and orf477 sequences were found respectively in the regions upstream and downstream of all blaCTX-M-15 genes. Seven different plasmid profiles were observed among the isolates. IncF (FII, FIA, FIB) and IncW replicons were identified in 11 CTX-M-15 producing isolates, and mostly, other replicons were also identified: IncHI2, IncA/C, IncP, IncI1 and IncX. All ESBL-producing E. coli isolates were integron positive and possessed "empty" integron structures with no inserted region of DNA. The following detected virulence genes were: (number of isolates in parentheses): fimA (ten); papC (seven); aer (five); eae (one); and papGIII, hly, cnf, and bfp (none). Molecular typing using pulsed-field gel electrophoresis and multilocus sequence typing showed a low genetic heterogeneity among the 12 ESBL-producing strains with five unrelated PFGE types and five different sequence types (STs) respectively. CTX-M-15-producing isolates were ascribed to phylogroup A (eleven isolates) and B2 (one isolate). CONCLUSION: To our knowledge, this study provides the first insight into the contribution of wild birds to the dynamics of ESBL-producing E. coli in Tunisia.

Species distribution, antibiotic resistance and virulence traits in canine and feline enterococci in Tunisia.

In order to investigate the possible role of dogs and cats in the carriage and potential dissemination of resistant enterococci, seventy faecal samples from dogs and cats were tested for enterococci. Fifty-eight enterococci were recovered. Isolates were identified as Enterococcus faecium (n = 31) and E. faecalis (n = 14) E. durans (n = 6), E. casseliflavus (n = 2), E. hirae and E. gallinarum (2 isolates each). Enterococcal isolates showed resistance to ciprofloxacin (n = 35), erythromycin (n = 31), tetracycline (n = 25), kanamycin (n = 15), streptomycin (n = 13), pristinamycin (n = 11), gentamicin (n = 10), chloramphenicol (n = 8), and linezolid (n = 6). The gene erm(B) was detected in 22 out of 31 erythromycin-resistant enterococci. All tetracycline-resistant enterococci carried tet(M) and/or tet(L) genes. The gene aac(6')-Ie-aph(2″)-Ia was identified in five of high-level gentamicin-resistant isolates, the genes aph(3')-IIIa and/or aac(6')-Ie-aph(2″)-Ia in eleven high-level kanamycin-resistant isolates and the gene ant(6)-Ia in eleven high-level streptomycin-resistant isolates. Only one strain harboured cat(A) gene, and five strains contained vat(E) or vat(D) genes. Virulence genes gel(E) (21 strains), esp (11 strains) and cylA/cylB (5 strains) were detected. High genetic diversity was demonstrated among E. faecium isolates by pulsed-field gel electrophoresis (PFGE). Dogs and cats can be carriers of antibiotic-resistant enterococci in their faeces that could shed into the household environment.

Prevalence, antimicrobial resistance and genetic lineages of Enterococcus spp. from vegetable food, soil and irrigation water in farm environments in Tunisia.

BACKGROUND: The objective of this study was to determine the species, clonal diversity, antibiotic resistance and virulence of enterococci in different environments. Seventy-one samples of farm origin (34 of food vegetables, 27 of soil and ten of irrigation water) and 19 samples of vegetables from five markets, were inoculated in Slanetz-Bartley agar plates supplemented or not with gentamicin (SB-Gen and SB plates, respectively) for enterococci recovery. RESULTS: Enterococci were obtained from 72.2% of tested samples in SB media (food vegetables from farms, 88.2%; soil and irrigation water, 51%; food vegetables from markets, 84.2%), and 65 enterococcal isolates were obtained. Enterococcus faecium was the most prevalent species (52.3%), followed by E. hirae (35.4%), E. faecalis (6.15%), and E. casseliflavus (6.15%). Antibiotic resistance detected among these enterococci was as follows (percentage/detected gene): ciprofloxacin (60%), erythromycin (18.4%/erm(B)), tetracycline (15.4%/tet(M)-tet(L)), kanamycin (15.4%/aph(3')-III), chloramphenicol (7.7%), streptomycin (3%/ant(6)), vancomycin (6.15%/vanC2)), teicoplanin (0%) and ampicillin (0%). High-level gentamicin-resistant (HLR-G) enterococci were detected in SB-Gen plates in 14 of 90 tested samples (15.5%), and 15 isolates were characterized: ten E. faecalis, four E. faecium and one E. hirae. All HLR-G enterococci carried the aac(6')-aph(2″), erm(B) and tet(M) genes, among other resistance genes. The HLR-G isolates showed high genetic diversity (ten different PFGE profiles), and were ascribed to the sequence types ST2, ST16, ST28 and new ST528 (in E. faecalis), and ST56, new ST885 and new ST886 (in E. faecium). CONCLUSION: Food vegetables in the farm or market settings are frequently contaminated by HLR-G enterococci, and these microorganisms could reach the human intestine through the food chain, if hygienic conditions are not followed. © 2015 Society of Chemical Industry.

Surveillance of Enterobacteriaceae from Diabetic Foot Infections in a Tunisian Hospital: Detection of E. coli-ST131-blaCTX-M-15 and K. pneumoniae-ST1-blaNDM-1 Strains.

The study determined the prevalence, antimicrobial resistant (AMR) determinants, and genetic characteristics of Escherichia coli and Klebsiella pneumoniae isolates from patients with diabetic foot infection (DFI) in a Tunisian hospital. A total of 26 Escherichia spp. and Klebsiella spp. isolates were recovered and identified by MALDI-TOF-MS. Antimicrobial susceptibility testing, the detection of AMR determinants and Shiga-like toxin genes, phylogenetic grouping, and molecular typing were performed. Twelve E. coli, 10 K. pneumoniae, 3 K. oxytoca, and 1 E. hermanii were isolated. A multidrug-resistant phenotype was detected in 65.4% of the isolates. About 30.8% of isolates were extended-spectrum ß-lactamase (ESBL) producers and mainly carried blaCTX-M-15 and blaCTX-M-14 genes. One blaNDM-1-producing K. pneumoniae-ST1 strain was identified. Class 1 integrons were detected in 11 isolates and 5 gene cassette arrangements were noted: dfrA1+aadA1 (n = 1), dfrA12+aadA2 (n = 3), and dfrA17+aadA5 (n = 1). Other non-ß-lactam resistance genes detected were as follows (number of isolates): aac(3')-II (3), aac(6')-Ib-cr(8), qnrB (2), qnrS (4), cmlA (2), floR (4), sul1 (11), sul2 (11), and sul3 (2). The phylogroup B1 was the most frequent (41.7%) among E. coli, and two ESBL-producing isolates corresponded to the ST131-B2 lineage. The ESBL- and carbapenemase-producing Enterobacteriaceae in DFIs are described for the first time in Tunisia.

Molecular characterization of Pseudomonas aeruginosa from diabetic foot infections in Tunisia.

Background. Pseudomonas aeruginosa is an invasive organism that frequently causes severe tissue damage in diabetic foot ulcers.Gap statement. The characterisation of P. aeruginosa strains isolated from diabetic foot infections has not been carried out in Tunisia.Purpose. The aim was to determine the prevalence of P. aeruginosa isolated from patients with diabetic foot infections (DFIs) in Tunisia and to characterize their resistance, virulence and molecular typing.Methods. Patients with DFIs admitted to the diabetes department of the International Hospital Centre of Tunisia, from September 2019 to April 2021, were included in this prospective study. P. aeruginosa were obtained from the wound swabs, aspiration and soft tissue biopsies during routine clinical care and were confirmed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. Antimicrobial susceptibility testing, serotyping, integron and OprD characterization, virulence, biofilm production, pigment quantification, elastase activity and molecular typing were analysed in all recovered P. aeruginosa isolates by phenotypic tests, specific PCRs, sequencing, pulsed-field gel electrophoresis (PFGE) and multilocus sequence typing.Results. Sixteen P. aeruginosa isolates (16.3 %) were recovered from 98 samples of 78 diabetic patients and were classified into 6 serotypes (O:11 the most frequent), 11 different PFGE patterns and 10 sequence types (three of them new ones). The high-risk clone ST235 was found in two isolates. The highest resistance percentages were observed to netilmicin (69 %) and cefepime (43.8 %). Four multidrug-resistant (MDR) isolates (25 %) were detected, three of them being carbapenem-resistant. The ST235-MDR strain harboured the In51 class 1 integron (intI1 +aadA6+orfD+qacED1-sul1). According to the detection of 14 genes involved in virulence or quorum sensing, 5 virulotypes were observed, including 5 exoU-positive, 9 exoS-positive and 2 exoU/exoS-positive strains. The lasR gene was truncated by ISPpu21 insertion sequence in one isolate, and a deletion of 64 bp in the rhlR gene was detected in the ST235-MDR strain. Low biofilm, pyoverdine and elastase production were detected in all P. aeruginosa; however, the lasR-truncated strain showed a chronic infection phenotype characterized by loss of serotype-specific antigenicity, high production of phenazines and high biofilm formation.Conclusions. Our study demonstrated for the first time the prevalence and the molecular characterization of P. aeruginosa strains from DFIs in Tunisia, showing a high genetic diversity, moderate antimicrobial resistance, but a high number of virulence-related traits, highlighting their pathological importance.

Antimicrobial resistance, virulence genes, and genetic lineages of Staphylococcus pseudintermedius in healthy dogs in tunisia.

Nasal swabs of 100 healthy dogs were obtained in 2011 in Tunisia and tested for Staphylococcus pseudintermedius recovery. Antimicrobial resistance profile and virulence gene content were determined. Multilocus-sequence-typing (MLST) and SmaI-pulsed-field gel electrophoresis (PFGE) were investigated. S. pseudintermedius was recovered in 55 of the 100 tested samples (55 %), and one isolate per sample was further studied. All 55 S. pseudintermedius isolates were susceptible to methicillin (MSSP) but showed resistance to the following antimicrobials (% resistant isolates/resistance gene): penicillin (56.4/blaZ), tetracycline (40/tetM), trimethoprim-sulfamethoxazole (23.7), fusidic acid (9), kanamycin (3.7/aph(3´)-Ia), erythromycin-clindamycin (1.8/erm(B)), streptomycin (1.8/ant(6)-Ia), chloramphenicol (1.8) and ciprofloxacin (1.8). The following toxin genes were identified (% of isolates): lukS/F-I (98.2), expA (5.5), se-int (98.2), sec canine (1.8), siet (100), sea (5.5), seb (3.6), sec (10.9), sed (54.5), sei (5.5), sej (29.1), sek (3.6), ser (9.1), and hlg v (38.2). Ten different sequence-types were detected among 11 representative MSSP isolates: ST20, ST44, ST69, ST70, ST78, ST100, ST108, ST160, ST161, and ST162, the last three ones revealing novel alleles or allele combinations. Eleven different PFGE-patterns were identified in these isolates. The nares of healthy dogs could be a reservoir of antimicrobial resistant and virulent MSSP, highlighting the presence of the recently described exfoliating gene expA and several enterotoxin genes.

Characterization of carbapenemase-producing Gram-negative bacilli: first report of blaNDM-1 in Enterobacter cloacae.

INTRODUCTION: The spread of multidrug-resistant bacteria, particularly carbapenem-resistant Gram-negative bacilli (CR-GNB), has become a serious challenge for clinicians due to limited therapeutic options. The aim of the study was to investigate the prevalence of carbapenemase production among clinical isolates recovered from 352 samples collected in Tebessa hospital, Algeria. METHODOLOGY: Bacterial isolates were identified by 16S RNA gene sequencing and susceptibility to antibiotics was determined by disk diffusion method. Carbapenem-resistant isolates were screened for carbapenemase production using modified carba Nordmann-Poirel test, modified Hodge test and imipenem-EDTA combined disc test. Extended-spectrum ß-lactamases (ESBL) were detected using double-disk synergy test. Molecular characterization of carbapenemases and ESBL genes was performed by polymerase chain reaction (PCR) and sequencing. RESULTS: A total of 85 Gram-negative bacilli isolates were recovered mainly from urine samples and were identified as: Klebsiella pneumoniae (17.65%), Serratia odorifera (15.29%), Escherichia coli (12.94%), Raoultella ornithinolytica, Enterobacter cloacae (11.76%), Serratia marcescens (10.59%), Morganella morganii (7.06%), Proteus mirabilis (5.88%), Acinetobacter baumannii (4.70%) and Pseudomonas aeruginosa (2.35%). All strains were resistant or intermediate to imipenem and/or ertapenem. ESBL, carbapenemase and metallo-beta-lactamases (MBL) phenotypes were detected in 19 (22.35%), 9 (10.59%) and 2 (2.35%) GNB isolates, respectively. PCR results in nine carbapenemase-producing GNB strains chosen showed the presence of one to four carbapenemase genes (blaGES, blaSME, blaNDM-1, blaVIM, blaGIM, blaSPM, blaOXA-48) in four strains; however, seven strains had at least one ESBL gene (blaTEM-1, blaCTXM-15, blaSHV). CONCLUSIONS: In this study, we report the first incidence of blaNDM-1 gene in Enterobacter cloacae isolated from urine sample in Algeria.

First Report of IMI-2-Producing Enterobacter bugandensis and CTX-M-55-Producing Escherichia coli isolated from Healthy Volunteers in Tunisia.

The aim of this study was to characterize the prevalence of fecal carriage of extended-spectrum beta-lactamases and carbapenemase-producing Gram-negative bacteria among healthy humans in Tunisia. Fifty-one rectal swabs of healthy volunteers were plated on MacConkey agar plates supplemented with cefotaxime or imipenem. The occurrences of resistance genes, integrons, and phylogroup typing were investigated using PCR and sequencing. The genetic relatedness of isolates was determined by pulsed-field-gel-electrophoresis (PFGE) and multilocus-sequence-typing (MLST). Whole-genome-sequencing (WGS) was performed for the carbapenem-resistant isolate. Sixteen ESBL-producing Escherichia coli isolates and one carbapenem-resistant Enterobacter bugandensis were detected out of the fifty-one fecal samples. The ESBL-producing E. coli strains contained genes encoding CTX-M-15 (n = 9), CTX-M-1 (n = 3), CTX-M-27 (n = 3), and CTX-M-55 (n = 1). Three CTX-M-1-producers were of lineages ST131, ST7366, and ST1158; two CTX-M-15-producers belonged to lineage ST925 and ST5100; one CTX-M-27-producer belonged to ST2887, and one CTX-M-15-producer belonged to ST744. Six isolates contained class 1 integrons with the following four gene cassette arrangements: dfrA5 (two isolates), dfrA12-orf-aadA2 (two isolates), dfrA17-aadA5 (one isolate), and aadA1 (one isolate). E. bugandensis belonged to ST1095, produced IMI-2 carbapenemase, and contained qnrE1 and fosA genes. A genome-sequence analysis of the E. bugandensis strain revealed new mutations in the blaACT and qnr genes. Our results reveal an alarming rate of ESBL-E. coli in healthy humans in Tunisia and the first description of IMI-2 in E. bugandensis.

High Prevalence of GES-5 Variant and Co-Expression of VIM-2 and GES-45 among Clinical Pseudomonas aeruginosa Strains in Tunisia.

Carbapenem-resistant Pseudomonas aeruginosa (CRPA) are a global health concern. The antimicrobial resistance, virulence, and molecular typing of 57 CRPA isolated from 43 patients who attended a specific Tunisian hospital from September 2018 to July 2019 were analyzed. All but one were multidrug-resistant CRPA, and 77% were difficult-to-treat-resistant (DTR) isolates. The blaVIM-2 gene was detected in four strains (6.9%), and among the 36 blaGES-positive CRPA (62%), the blaGES-5 gene was the predominant variant (86%). Three strains co-harbored the blaVIM-2 and blaGES-45 genes, and seven CRPA carried the blaSHV-2a gene (14%). OprD alterations, including truncations by insertion sequences, were observed in 18 strains. Regarding the 46 class 1 integron-positive CRPA (81%), the blaGES-5 gene was located in integron In717, while the blaGES-29 and blaGES-45 genes were found in two new integrons (In2122 and In4879), and the blaVIM-2 gene was found in In1183 and the new integron In2142. Twenty-four PFGE patterns and thirteen sequence types (three new ones) were identified. The predominant serotype O:11 and exoU (81%) were mostly associated with ST235 and the new ST3385 clones. The seven blaSHV-2a-CRPA from different patients belonged to ST3385 and the same PFGE pattern. The blaGES-5- and blaVIM-2 + blaGES-45-positive CRPA recovered mostly from ICU patients belonged to the high-risk clone ST235. Our results highlight the alarming prevalence of blaGES-5- and ST235-CRPA, the co-existence of blaGES-45 and blaVIM-2, and their location within integrons favoring their dissemination.

High diversity of genetic lineages and virulence genes in nasal Staphylococcus aureus isolates from donkeys destined to food consumption in Tunisia with predominance of the ruminant associated CC133 lineage.

BACKGROUND: The objective of this study was to determine the genetic lineages and the incidence of antibiotic resistance and virulence determinants of nasal Staphylococcus aureus isolates of healthy donkeys destined to food consumption in Tunisia. RESULTS: Nasal swabs of 100 donkeys obtained in a large slaughterhouse in 2010 were inoculated in specific media for S. aureus and methicillin-resistant S. aureus (MRSA) recovery. S. aureus was obtained in 50% of the samples, being all of isolates methicillin-susceptible (MSSA). Genetic lineages, toxin gene profile, and antibiotic resistance mechanisms were determined in recovered isolates. Twenty-five different spa-types were detected among the 50 MSSA with 9 novel spa-types. S. aureus isolates were ascribed to agr type I (37 isolates), III (7), II (4), and IV (2). Sixteen different sequence-types (STs) were revealed by MLST, with seven new ones. STs belonging to clonal clomplex CC133 were majority. The gene tst was detected in 6 isolates and the gene etb in one isolate. Different combinations of enterotoxin, leukocidin and haemolysin genes were identified among S. aureus isolates. The egc-cluster-like and an incomplete egc-cluster-like were detected. Isolates resistant to penicillin, erythromycin, fusidic acid, streptomycin, ciprofloxacin, clindamycin, tetracycline, or chloramphenicol were found and the genes blaZ, erm(A), erm(C), tet(M), fusC were identified. CONCLUSIONS: The nares of donkeys frequently harbor MSSA. They could be reservoirs of the ruminant-associated CC133 lineage and of toxin genes encoding TSST-1 and other virulence traits with potential implications in public health. CC133 seems to have a broader host distribution than expected.

Prevalence and characterization of extended-spectrum beta-lactamase (ESBL)- and CMY-2-producing Escherichia coli isolates from healthy food-producing animals in Tunisia.

The prevalence of extended-spectrum beta-lactamase (ESBL)- and plasmidic AmpC-beta-lactamase (pAmpC-BL)-producing Escherichia coli isolates has been studied in food-producing animals at the farm level in Tunisia, and recovered isolates were characterized for the presence of other resistance genes and integrons. Eighty fecal samples of food-producing animals (23 sheep, 22 chickens, 22 cattle, six horses, five rabbits, and two dromedaries) were obtained from 35 different farms in Tunisia in 2011. Samples were inoculated onto MacConkey agar plates supplemented with cefotaxime (2 mg/L) for cefotaxime-resistant (CTX(R)) E. coli recovery. CTX(R) E. coli isolates were detected in 11 out of 80 samples (13.8%), and one isolate per sample was further characterized (10 from chickens and one from a dromedary). The 11 CTX(R) isolates were distributed into phylogroups: B1 (five isolates), A (two isolates), D (three isolates), and B2 (one isolate). The following beta-lactamase genes were detected: bla(CTX-M-1) (seven isolates), bla(CTX-M-1)+bla(TEM-135) (one isolate), bla(CTX-M-1)+bla(TEM-1b) (one isolate), and bla(CMY-2) (two isolates). All ESBL- and pAmpC-BL-producing E. coli strains showed unrelated pulsed-field gel electrophoresis patterns. Seven isolates contained class 1 integrons with four gene cassette arrangements: dfrA17-aadA5 (three isolates), dfrA1-aadA1 (two isolates), dfrA15-aadA1 (one isolate), and aadA1 (one isolate). All isolates showed tetracycline resistance and contained the tet(A) +/- tet(B) genes. Virulence genes detected were as follows (number of isolates in parentheses): fimA (10); aer (eight); papC (two); and papGIII, hly, cnf, and bfp (none). Chicken farms constitute a reservoir of ESBL- and pAmpC-BL-producing E. coli isolates of the CTX-M-1 and CMY-2 types that potentially could be transmitted to humans via the food chain or by direct contact.

Antimicrobial activity and safety features assessment of Weissella spp. from environmental sources.

Weissella strains have been reported to be useful in biotechnological and probiotic determinations, and some of them are considered opportunistic pathogens. Given the widespread interest about antimicrobial susceptibilities, transmission of resistances, and virulence factors, there is little research available on such topics for Weissella. The aim of this study was to assess the safety aspects and antimicrobial potential of 54 Weissella spp. strains from different environmental sources. Antibiotic susceptibility, hemolytic activity, horizontal transfer, and antibacterial activity were studied, as well as the detection of biogenic amine BA production on decarboxylase medium and PCR was performed. All the strains were nonhemolytic and sensitive to chloramphenicol and ampicillin. Several strains were classified as resistant to fusidic acid, and very low resistance rates were detected to ciprofloxacin, tetracycline, streptomycin, lincomycin, erythromycin, and rifampicin, although all strains had intrinsic resistance to vancomycin, nalidixic acid, kanamycin, and teicoplanin. Two BA-producing strains (W. halotolerans FAS30 and FAS29) exhibited tyrosine decarboxylase activity, and just one W. confusa FS077 produced both tyramine and histamine, and their genetic determinants were identified. Ornithine decarboxylase/odc gene was found in 16 of the Weissella strains, although 3 of them synthesize putrescine. Interestingly, eight strains with good properties displayed antibacterial activity. Conjugation frequencies of erythromycin from Bacillus to Weissella spp. varied in the average of 3 × 10-9 transconjugants/recipient. However, no tetracycline-resistant transconjugant was obtained with Enterococcus faecalis JH2-2 as recipient. The obtained results support the safe status of Weissella strains, derived from environmental sources, when used as probiotics in animal feed.

Fungal Community Investigation from Propolis Natural Products: Diversity and Antibacterial Activities Evaluation.

Discovering new species and interesting bioactive metabolites from customary sources is becoming progressively laborious. Propolis constitutes the largest diversified reserve of microbial constituents in the beehive. However, fungal communities associated with these environments remain insufficiently established. We present the first detailed investigation of the cultivable fungal community associated with Tunisian propolis, and we evaluate its antibacterial properties against pathogenic bacteria. A total of 80 fungal strains were isolated from propolis samples derived from seven different Tunisian locations. The majority of the isolated fungi were classified as Ascomycota (97.5%), and only 2.5% belonged to Basidiomycota. Our collection was clustered into 15 genera, among which Coniochaeta (36.25%), Aspergillus (15%), Penicillium (13.75%), Cladosporium (10%), Fusarium (7.5%), Didymella (5%), and Alternaria (3.75%) were the most common. Evaluation of the antibacterial activity revealed that 25.6% of the total community showed a broad range of antibacterial activity. Particularly, the Penicillium griseofulvum CC8 strain has manifested the strongest inhibitory effects against all the tested bacteria.

Whole Genome Sequencing of a Citrobacter freundii Strain Isolated from the Hospital Environment: An Extremely Multiresistant NDM-1 and VIM-48 Coproducing Isolate.

Citrobacter freundii has acquired resistance to several antimicrobial drugs, including last-resort antibiotics affecting, therefore, clinical efficacy and causing high rates of mortality. In this study, we investigate the whole genome sequence of a carbapenem-resistant C. freundii strain isolated from the hospital environment in Tunisia. A total of 210 samples were taken using sterile swabs, from inanimate surfaces, medical devices, and care staff, during the period extended between March and April 2019. After the microbiological analysis of samples and antimicrobial susceptibility testing, only one strain identified as C. freundii showing resistance to carbapenems was selected for the whole genome sequencing. The genome analysis revealed a high-level resistance to most antibiotics. Interestingly, we have noted the coexistence of blaNDM-1 and blaVIM-48 metallo-ß-lactamase (MBL) encoding genes conferring resistance to carbapenems. Other ß-lactamases encoding genes have also been detected, including blaTEM-1, blaCMY-48, and blaOXA-1. Moreover, genes conferring resistance to aminoglycoside [aac(3)-IId, ant(3″)-Ia, aadA, aac(6')-Ib], macrolide [mph(A)], sulfonamide (sul1), trimethoprim (dfrA1), tetracycline [tet(D)], chloramphenicol [cat(B3)], rifamycin (arr-3), and quinolone (qnrB) have been revealed. The multi-locus sequence typing analysis showed that this isolate could not be assigned to an existing sequence type (ST), but it is almost identical to ST22. The plasmid investigation revealed the presence of five plasmids belonging to diverse incompatibility groups (IncFII, IncHI1A, IncHI1B, IncN, and IncX3). To the best of our knowledge, our findings report the first detection of NDM-1 and VIM-48 coproducing C. freundii in Tunisia and the second detection in the world of the blaVIM-48.

Methicillin-Resistant Staphylococcus aureus from Diabetic Foot Infections in a Tunisian Hospital with the First Detection of MSSA CC398-t571.

This study sought to analyze the antimicrobial resistant phenotypes and genotypes as well as the virulence content of S. aureus isolates recovered from patients with diabetic foot infections (DFIs) in a Tunisian hospital. Eighty-three clinical samples of 64 patients were analyzed, and bacterial isolates were identified by MALDI-TOF. The antimicrobial resistance phenotypes were determined by the Kirby-Bauer disk diffusion susceptibility test. Resistance and virulence genes, agr profile, spa and SCCmec types were determined by PCR and sequencing. S. aureus was detected in 14 of the 64 patients (21.9%), and 15 S. aureus isolates were recovered. Six out of the fifteen S. aureus isolates were methicillin-resistant (MRSA, mecA-positive) (40%). The isolates harbored the following resistance genes (number of isolates): blaZ (12), erm(B) (2), erm(A) (1), msrA (2), tet(M) (2), tet(K) (3), tet(L) (1), aac(6')-aph(2″) (2), ant(4″) (1) and fexA (1). The lukS/F-PV and tst genes were detected in three isolates. Twelve different spa-types were identified and assigned to seven clonal complexes with the predominance of agr-type III. Furthermore, the SCCmec types III, IV and V were found among the MRSA isolates. Moreover, one MSSA CC398-t571-agr-III isolate was found; it was susceptible to all antimicrobial agents and lacked luk-S/F-PV, tst, eta and etb genes. This is the first report on the prevalence and molecular characterization of S. aureus from DFIs and also the first detection of the MSSA-CC398-t571 clone in human infections in Tunisia. Our findings indicated a high prevalence S. aureus in DFIs with genetic diversity among the MSSA and MRSA isolates.

Efficiency of a Tetracycline-Adjuvant Combination Against Multidrug Resistant Pseudomonas aeruginosa Tunisian Clinical Isolates.

The growing number of multidrug resistant strains in Tunisia has become a serious health concern contributing to high rate of mortality and morbidity. Since current antibiotics are rapidly becoming ineffective, novel strategies to combat resistance are needed. Recently, we demonstrated that combination of a tetracycline antibiotic with various polyaminoisoprenyl adjuvants can sustain the life span and enhance the activity of these drugs against Pseudomonas aeruginosa reference strain (PA01). In the context of our continuing studies, the effective approach of antibiotic-adjuvant was investigated against a large panel of P. aeruginosa Tunisian clinical strains collected from the Military Hospital of Tunis. In this paper, we demonstrated that the combination of a farnesyl spermine compound 3 used at concentrations ranging from 2.5 to 10 µM, in the presence of doxycycline or minocycline leads to a significant decrease of P. aeruginosa antibiotic resistance.