Lineages, Virulence Gene Associated and Integrons among Extended Spectrum ß-Lactamase (ESBL) and CMY-2 Producing Enterobacteriaceae from Bovine Mastitis, in Tunisia.

Extended Spectrum Beta-Lactamase (ESBL) Enterobacteriaceae are becoming widespread enzymes in food-producing animals worldwide. Escherichia coli and Klebseilla pneumoniae are two of the most significant pathogens causing mastitis. Our study focused on the characterization of the genetic support of ESBL/pAmpC and antibiotic resistance mechanisms in cefotaxime-resistant (CTXR) and susceptible (CTXS) Enterobacteriaceae isolates, recovered from bovine mastitis in Tunisia, as well as the analyses of their clonal lineage and virulence-associated genes. The study was carried out on 17 ESBL/pAmpC E. coli and K. pneumoniae and 50 CTXS E. coli. Detection of resistance genes and clonal diversity was performed by PCR amplification and sequencing. The following ß-lactamase genes were detected: blaCTX-M-15 (n = 6), blaCTX-M-15 + blaOXA-1 (2), bla CTX-M-15 + blaOXA-1 + blaTEM-1b (2), blaCTX-M-15 + blaTEM-1b (4), blaCMY-2 (3). The MLST showed the following STs: ST405 (n = 4 strains); ST58 (n = 3); ST155 (n = 3); ST471 (n = 2); and ST101 (n = 2). ST399 (n = 1) and ST617 (n = 1) were identified in p(AmpC) E. coli producer strains. The phylogroups A and B1 were the most detected ones, followed by the pathogenic phylogroup B2 that harbored the shigatoxin genes stx1/stx2, associated with the cnf, fimA, and aer virulence factors. The qnrA/qnrB, aac(6')-Ib-cr genes and integrons class 1 with different gene cassettes were detected amongst these CTXR/S isolated strains. The presence of different genetic lineages, associated with resistance and virulence genes in pathogenic bacteria in dairy farms, may complicate antibiotic therapies and pose a potential risk to public health.

First Detection of Human ST131-CTX-M-15-O25-B2 Clone and High-Risk Clonal Lineages of ESBL/pAmpC-Producing E. coli Isolates from Diarrheic Poultry in Tunisia.

Circulation of a multi-resistance clone of bacteria associated with genetic elements in diseased animals constitutes a global public health problem. Our study focused on the characterization of the support of ESBL in cefotaxime resistant E. coli (CTXR) isolates recovered from poultry with diarrhea, analysis of their clonal lineage, and virulence-associated genes. The study was carried out on 130 samples of chickens with diarrhea, collected in 2015 from poultry farms in Tunisia. Isolates of 20 CTXR E. coli strains were identified as ESBL and AmpC ß- lactamase producers. The following ß-lactamase genes (number of isolates) were detected: blaCTX-M-15+ blaOXA1 (4), blaCTX-M-15 + blaOXA1 + blaTEM-1b (2), blaCTX-M-1 + blaTEM-1b (9), blaCTX-M-1 (2), blaCMY2 + blaTEM-1b (3). Six E. coli harboring blaCTXM-15 were allocated to ST131-B2-O25b-; six and three blaCTX-M-1 were grouped in ST155, ST10, and ST58, respectively, related to the phylogroup D and A. The qnrB gene, the variant aac(6')-Ib-cr, and the class 1 integrons with different gene cassettes, were detected amongst our 20 isolated strains, which were classified as ExPEC and aEPEC. Our findings highlighted the emergence of the human pandemic ST131-CTX-M-15-O25-B2 clone and the high risk of such clonal lineage strains in diarrheic poultry, in Tunisia, which could constitute a risk of their transfer to healthy animals and humans.

Epidemiology of ß-Lactamase-Producing Staphylococci and Gram Negative Bacteria as Cause of Clinical Bovine Mastitis in Tunisia.

The aim of this study was to determine the species distribution of Staphylococcus, Gram negative bacteria (GNB) and the occurrence of Methicillin Resistant Staphylococci (MRS) and Extended-Spectrum ß-lactamase- (ESBL-) producing GNB. Bacterial culture of 300 clinical mastitis milk samples from 30 different farms across different regions of Tunisia during four seasons was realized. The obtained results showed the presence of high frequency of the tested samples with a positive growth for bacteria (64%). In addition a high recovery rate of Staphylococci and/or GNB in these clinical mastitis milk samples (87%) was detected. In addition, a high percentage of GNB (68.2%) compared to Staphylococcus species (32%) was noted. Moreover, a significant variation of the number of these bacteria according to the farm location, the seasons, and cows age was detected. The highest percentage was observed in the North of Tunisia during the winter and the spring seasons in adult cows with a dominance of GNB growth. Coagulase negative Staphylococci (CNS) (n=11) and GNB (n=16) species were identified. Escherichia coli (E. coli) was the most frequently found bacterium followed by Klebsiella pneumoniae. The dominant Staphylococcus isolates was S. xylosus followed by S. aureus the major pathogen isolated. Methicillin resistance was confirmed by the presence of the mecA gene in 3 S. aureus and 14 CNS isolates; all of these isolates were lacking the mecC gene. Various species of GNB, resistant to cefotaxime, were detected (n=15). ESBLs were detected on selective medium in 10 E. coli and 4 K. pneumoniae. All ESBL producers strains carry the blaCTX-M. The presence of different resistant mastitis pathogens in dairy farms may complicate therapeutic options and contaminated animals could become zoonotic agent reservoir for human.

Detection and characterization of methicillin-resistant and susceptible coagulase-negative staphylococci in milk from cows with clinical mastitis in Tunisia.

OBJECTIVES: This study investigated prevalence of methicillin-resistant (MR) and methicillin-susceptible (MS) coagulase-negative staphylococci (CNS) and the implicated mechanisms of resistance and virulence in milk of mastitis cows. In addition, the presence of SCCmec type was analyzed in MR Staphylococcus epidermidis (MRSE). RESULTS: Three hundred milk samples from cows with clinical mastitis were obtained from 30 dairy farms in different regions of Tunisia. Sixty-eight of the 300 tested samples contained CNS strains. Various CNS species were identified, with Staphylococcus xylosus being the most frequently found (40%) followed by Staphylococcus warneri (12%). The mecA gene was present in 14 of 20 MR-CNS isolates. All of them were lacking the mecC gene. The SCCmecIVa was identified in four MRSE isolates. Most of CNS isolates showed penicillin resistance (70.6%) and 58.3% of them carried the blaZ gene. MR-CNS isolates (n = 20) showed resistance to erythromycin, tetracycline and trimethoprim-sulfametoxazole harboring different resistance genes such us erm(B), erm(T), erm(C), mph(C) or msr(A), tet(K) and dfr(A). However, a lower percentage of resistance was observed among 48 MS-CNS isolates: erythromycin (8.3%), tetracycline (6.2%), streptomycin (6.2%), clindamycin (6.2%), and trimethoprim-sulfametoxazole (2%). The Inu(B) gene was detected in one Staphylococcus xylosus strain that showed clindamycin resistance. The virulence gene tsst-1 was observed in one MR-CNS strain. DISCUSSION: Coagulase-negative staphylococci containing a diversity of antimicrobial resistance genes are frequently detected in milk of mastitis cows. This fact emphasizes the importance of identifying CNS when an intramammary infection is present because of the potential risk of lateral transfer of resistant genes among staphylococcal species and other pathogenic bacteria.

Molecular Characterization and Clonal Diversity of Methicillin-Resistant and -Susceptible Staphylococcus aureus Isolates of Milk of Cows with Clinical Mastitis in Tunisia.

The aim of this study was to determine the genetic lineages, and the frequency of antibiotic resistance and virulence determinants in methicillin-resistant Staphylococcus aureus (MRSA) and methicillin-susceptible S. aureus (MSSA) isolates recovered from milk of cows with clinical mastitis. Three hundred milk samples from bovine with clinical mastitis were obtained from 30 dairy farms in different regions of Tunisia. Fifteen of the 300 tested samples contained S. aureus (5%), in three cases were MRSA. Isolates (one/sample) were typed (S. aureus protein A [spa], multilocus sequence typing and accessory gene regulator [agr]). The presence of resistance and virulence genes was analyzed by PCR. The three MRSA isolates contained mecA and blaZ genes (one of them also the msr(A) gene), and carried the enterotoxin gene sen; they were typed as t10381-ST4114 or t267-ST4120, and corresponded to agr type-I. Twelve MSSA isolates were recovered and harbored the blaZ (7 strains) or erm(C) genes (1 strain). The MSSA isolates presented seven different spa-types, associated to new sequence types (STs): t426-ST4118, t267-ST4120, t1773-ST4115, t509-ST4119, t529-ST4117, t2844-ST4113, and t2802-ST4112; most isolates (8/12) were typed as t267/ST4120. All S. aureus isolates were scn-negative, except one MSSA of lineage ST4119 that exhibited the immune evasion cluster type D, and harbored the seg, sei, sem, seo, and seu enterotoxin genes. Four MSSA isolates carried the toxic shock syndrome toxin 1 gene (tst). S. aureus (including MRSA) is an important cause of bovine mastitis, showing isolates with high genetic diversity and high content in virulence genes.