Genetic background of aminoglycoside-modifying enzymes in various genetic lineages of clinical aminoglycosides-resistant E. coli and K. pneumoniae isolates in Tunisia.

AIMS: This study was conducted to evaluate the in vitro activity of clinically relevant aminoglycosides and to determine the prevalence of genes encoding aminoglycoside modifying enzymes (AMEs) and 16S ribosomal RNA (rRNA) methyltransferases among aminoglycoside-resistant E. coli (n = 61) and K. pneumoniae (n = 44) clinical isolates. Associated resistances to beta-lactams and their bla genes as well as the genetic relatedness of isolates were also investigated. MATERIALS AND METHODS: A total of 105 aminoglycoside-resistant E. coli (n = 61) and K. pneumoniae (n = 44) isolates recovered between March and May 2017 from 100 patients hospitalized in different wards of Charles Nicolle Hospital of Tunis, Tunisia, were studied. Minimal inhibitory concentrations of aminoglycoside compounds were determined by broth microdilution method. Aminoglycosides resistance encoding genes [aph(3´)-Ia, aph(3') IIa, aph(3´)-VIa, ant(2″)-Ia, aac(3)-IIa, aac(3)-IVa, aac(6')-Ib, rmtA, rmtB, rmtC, armA, and npmA] and bla genes were investigated by PCR and sequencing. Genetic relatedness was examined by multilocus sequence typing (MLST) for representative isolates. RESULTS: High rates of aminoglycoside resistance were found: gentamicin (85.7%), tobramycin (87.6%), kanamycin (78.0%), netilmincin (74.3%), and amikcin (18.0%). Most common AME gene was aac(3)-IIa (42%), followed by aac(6')-Ib (36.2%) and aph(3')-VIa (32.4%). The majority of isolates were resistant to beta-lactams and blaCTX-M-15 was the most common ESBL. The blaNDM-1 and blaOXA-48 were also produced by 1 and 23 isolates, respectively. Novel sequence types have been reported among our isolates and high-risk clonal lineages have been detected, such as E. coli ST43 (ST131 in Achtman MLST scheme) and K. pneumoniae (ST11/ST13). CONCLUSIONS: The high prevalence of aminoglycoside resistance rates and the diversity of corresponding genes, with diverse ß-lactamase enzymes among genetically heterogeneous clinical isolates present a matter of concern.

Genetic characterization of vancomycin-resistant Enterococcus faecium isolates from neutropenic patients in Tunisia: spread of the pandemic CC17 clone associated with high genetic diversity in Tn1546-like structures.

AIMS: In Tunisia, limited research has focused on characterizing clinical vancomycin-resistant Enterococcus faecium (VREfm). This study aimed to bridge this knowledge gap by molecular characterization of antimicrobial resistance, determining the genetic elements mediating vancomycin-resistance, and whole-genome sequencing of one representative VREfm isolate. METHODS AND RESULTS: Over 6 years (2011-2016), a total of eighty VREfm isolates responsible for infection or colonization were identified from hospitalized patients, with the incidence rate increasing from 2% in 2011 to 27% in 2016. All of these strains harbored the vanA gene. The screening for antimicrobial resistance genes revealed the predominance of ermB, tetM, and aac(6')-Ie-aph(2'')-Ia genes and 81.2% of strains harbored the Tn1545. Pulsed-field gel electrophoresis identified seven clusters, with two major clusters (belonging to ST117 and ST80) persisting throughout the study period. Seven Tn1546 types were detected, with type VI (truncated transposon) being the most prevalent (57.5%). Whole-genome sequencing revealed a 3 028 373 bp chromosome and five plasmids. Mobile genetic elements and a type I CRISPR-cas locus were identified. Notably, the vanA gene was carried by the classic Tn1546 transposon with ISL3 insertion on a rep17pRUM plasmid. CONCLUSION: A concerning trend in the prevalence of VREfm essentially attributed to CC17 persistence and to horizontal transfer of multiple genetic variants of truncated vanA-Tn1546.

Outbreak caused by pandrug-resistant blaOXA-69/blaOXA-23/blaGES harboring Acinetobacter baumannii ST2 in an intensive care unit.

Acinetobacter baumannii has emerged as a main nosocomial pathogen exhibiting high rates of resistance to clinically relevant antibiotics. Six pandrug-resistant A. baumannii (PDR-A. baumannii) were recovered from three patients in a Tunisian Intensive Care Unit (ICU) between 10th and 16th of May 2018 resulting in one fatal case and raising the possibility of an outbreak. On 18th of May environmental screening of ICU surfaces was carried out. On 22nd of May a fourth patient was infected with PDR-A. baumannii and died. A second investigation was carried out for environmental screening and PDR-A. baumannii was isolated from the respirator. Antimicrobial susceptibility testing was performed according to EUCAST (2019) guidelines. MIC of colistin was determined by broth microdilution method. PCR was used to detect 14 beta-lactamases/carbapenemases and mcr (mcr-1 to mcr-5) genes. The genetic relatedness of PDR-A. baumannii isolates was determined by PFGE and MLST. Seven PDR-A. baumannii isolates were recovered from four patients, one MDR strain from wash basin, a PDR strain from hand sanitizer bottle and another PDR strain from respirator. All PDR-A. baumannii (n = 9) harbored blaOXA-69 gene and none carried mcr. Moreover, seven carried blaGES and blaOXA-23 genes. PFGE identified four pulsotypes (A, B, C, and D) with the pulsotype A gathering seven PDR-A. baumannii isolates: six from three patients and one from hygiene sample. MLST revealed that all PDR-A. baumannii isolates of pulsotype A belonged to the pandemic clone ST2. Systematic screening of MDR and PDR-A. baumannii is highly recommended to limit dissemination of such strains in ICUs.

Occurrence of virulence genes and methicillin-resistance in Staphylococcus aureus isolates causing subclinical bovine mastitis in Tiaret area, Algeria.

Mastitis remains the most frequent and the most expensive disease of dairy breeding. The objective of the study was to study S. aureus isolates collected from subclinical bovine mastitis in the Tiaret region, Algeria, by determining their antimicrobial susceptibility and their virulence traits. Sixty-two S. aureus isolates collected from subclinical bovine mastitis were studied by determining their antimicrobial susceptibility according to CLSI guidelines, and nine genes encoding virulence factors and resistance to methicillin and penicillin were determined by PCR. Multi-drug resistance was observed in 19 (30.64%) isolates and five (8%) were methicillin-resistant S. aureus (MRSA), four of them harbored the mecA gene; however, the mecC gene was not detected. Out of 59 penicillin-resistant isolates, 14 harbored the blaZ gene; one of them co-harbored the mecA gene. The following virulence genes were detected: eta (n = 23; 37%), icaA (20; 32.2%), icaD (18; 29%), etb (16; 25.8%), luk E-D (14; 22.5%), and sea (6; 9.6%). The tsst-1, lukF/lukS, and luk-M genes were not detected. The occurrence of MRSA and multi-drug resistant (MDR) S. aureus isolates as well as genes encoding virulence factors playing an important role in the pathogenesis of subclinical bovine mastitis and of harmful potential to human is a cause for concern.

Widespread of the Vienna/Hungarian/Brazilian CC8-ST239-SCCmec III MRSA clone in patients hospitalized in the Tunisian Burn and Traumatology Center.

The emergence and spread of methicillin-resistant Staphylococcus aureus (MRSA) in hospitals is a major global public health concern. The current study sought to characterize 25 MRSA clinical isolates collected in a Tunisian hospital from December 2015 to September 2016, with the genetic lineages, virulence factors, and antibiotic resistance mechanisms determined for these isolates. Three spa-types were detected: t037 (23 isolates), t932, and t2235 (one isolate each). Isolates were ascribed to agr I (n = 20), agr II (n = 1), with four nontypeable isolates. Depending on sequence type (ST), the 25 MRSA isolates were assigned to two clonal complexes (CC8 and CC5), with a predominance of the lineage ST239-CC8 (n = 24; 96%). All isolates belonging to CC8 had the SCCmec type III, while the unique CC5 isolate had SCCmec type IV. Antimicrobial susceptibility testing revealed high levels of resistance to aminoglycosides, tetracycline, ciprofloxacin and rifampicin for the majority of isolates belonging to the ST239-CC8 lineage. The ST149-CC5 isolate was susceptible to non-ß-lactam antibiotics. One isolate harbored the tsst-1 gene (4%); however, lukS/LukF-PV, eta and etb genes were not detected. The MDR ST239-CC8 clone would seem to be widespread in this hospital. Therefore, a rigorous hygienic control system is urgently required.

Detection of carbapenem resistant Pseudomonas aeruginosa co-harboring blaVIM-2 and blaGES-5 in burn patients.

Pseudomonas aeruginosa is one of the major infectious agents in burn patients. Globally, high rates of antimicrobial resistance in P. aeruginosa have been reported, which is a cause of concern. The objective of this study was to determine the rate of resistance to carbapenems in P. aeruginosa isolates recovered from burn patients in Tunisia, to search genes encoding for carbapenemases and to determine their epidemiological markers (serotypes). A retrospective study was conducted in the Burn Intensive Care Unit (BICU) of the Trauma and Burn Centre of Ben Arous, Tunisia, and P. aeruginosa isolates collected from burn patients, from January to December 2018 were investigated. Carbapenemase screening was performed by Carbapenem Inactivation Method (CIM) and by EDTA-disk test for all carbapenem resistant isolates. Genes encoding carbapenemases (blaVIM, blaIMP, blaGES, blaNDM, and blaKPC) were investigated by PCR and selected carbapenemase genes were sequenced. During the study period, 104 non duplicated P. aeruginosa isolates were recovered. Most of them were isolated from skin samples (45.1%) and blood culture (22.1%) and belonged to O:11 (19.2%), O:12, and O:5 (12.5%, each) serotypes. High rates of resistance were observed for carbapenems (64.4%). Among the 67 carbapenem resistant isolates, 58 (86.5%) harbored blaVIM gene and 55 (82%) blaGES gene; in addition, 48 (71.6%) co-harbored blaVIM and blaGES genes. After sequencing, the blaVIM-2 and blaGES-5 gene variants were identified in seven randomly selected isolates. To the best of our knowledge, this is the first description of P. aeruginosa simultaneously harboring blaVIM-2 and blaGES-5 genes.

High-risk clonal lineages among extended-spectrum ß-lactamase producing Escherichia coli and Klebsiella pneumoniae from urban and rural stagnant water samples in Tunisia.

This study sought to investigate the occurrence and subsequently to characterize extended-spectrum beta-lactamase (ESBL)-producing Enterobacteriaceae from urban and rural stagnant water samples during the wet season (December to February) in several regions of northern Tunisia. From 56 stagnant water samples, 14 ESBL-producing Enterobacteriaceae were recovered, including 9 Escherichia coli, 3 Klebsiella pneumoniae, and 2 K. oxytoca. Most isolates were multidrug-resistant, with ESBL production primarily encoded by blaCTX-M-15 (n = 8) and blaCTX-M-1 (n = 4) followed by blaCTX-M-55 (n = 1) and blaTEM-26 (n = 1). One K. pneumoniae isolate co-harbored blaKPC and blaCTX-M-15 genes. Class 1 integrons were detected in 4 isolates, however, sul1, sul2, and aac(6')-Ib-cr genes were detected in eleven, two, and four isolates, respectively. The nine E. coli isolates belonged to seven sequence types namely, B2/ST131 (3 isolates), A/ST164, A/ST10, A/ST224, A/ST38, A/ST155, and A/ST69 (each of them one isolate). The three K. pneumoniae isolates were assigned to three sequence types: ST101, ST405 (harboring CTX-M-15 and KPC), and ST1564. Overall, the phenotypic and genotypic traits of collected isolates mirror the molecular epidemiology of ESBL-producing enterobacteria in Tunisia and highlight the potential role of stagnant water in both urban and rural areas as a reservoir of ESBL-producing Enterobacteriaceae.

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.

Identification and characterization of multidrug-resistant ESBL-producing Salmonella enterica serovars Kentucky and Typhimurium isolated in Tunisia CTX-M-61/TEM-34, a novel cefotaxime-hydrolysing ß-lactamase of Salmonella.

AIMS: Molecular characterization of extended-spectrum ß-lactamases (ESBLs) among Salmonella Kentucky and Typhimurium isolates: partial sequence analysis of the types of ß-lactamases found in these isolates, clonality, resistance and supposed emergence of ESBL-producing strains. METHODS AND RESULTS: A retrospective study surveyed the ESBLs occurring in a total of 1404 Salmonella Kentucky and Typhimurium isolates collected over a 5-year period in Tunisia. Antimicrobial susceptibility tests, ESBL phenotype determination (double-disc synergy) were performed. Polymerase chain reaction assays were used for the detection of ß-lactamase genes (blaTEM , blaSHV , blaOXA-1 and blaCTX-M ), class 1 and class 2 integrases (intI1 and intI2) and the 3' conserved segment (3'-CS) of class 1 integron (qacEΔ1+sul1). Sequencing of amplicons of ß-lactamase genes was performed. Percentage of 9.8 of the isolates (S. Kentucky = 117, S. Typhimurium = 20) were either resistant to penicillin and had decreased susceptibility to cefotaxime or had a positive double-disc synergy test result. Polymerase chain reaction detected that these isolates harboured one or more ß-lactamase genes (blaTEM , blaSHV , blaOXA-1 or blaCTX-M ). TEM-1, TEM-34, CTX-M15, CTX-M9 and CTX-M61 type ESBLs were identified through sequencing. The novel Salmonella cefotaxime-hydrolysing ß-lactamase, CTX-M61/TEM-34, detected in this study showed the emergence of new CTX-M-type ESBLs in Tunisia. There were found 33 different multidrug resistance (MDR) patterns. CONCLUSION: These findings highlighted the proliferation of ESBLs and MDR in Salmonella Kentucky and Typhimurium isolates from numerous regions and sources in Tunisia, indicating an emerging public health concern. SIGNIFICANCE AND IMPACT OF THE STUDY: For the first time CTX-M-61/TEM-34, a novel cefotaxime-hydrolysing ß-lactamase of Salmonella had been detected.

Study of the diversity of 16S-23S rDNA internal transcribed spacer (ITS) typing of Escherichia coli strains isolated from various biotopes in Tunisia.

We investigated the 16S-23S rRNA intergenic spacer region (ISR)-PCR and the phylogenetic PCR analyses of 150 Escherichia coli isolates as tools to explore their diversity, according to their sampling origins, and their relative dominance in these sampling sources. These genetic markers are used to explore phylogenetic and genetic relationships of these 150 E. coli isolates recovered from different environmental sources (water, food, animal, human and vegetables). These isolates are tested for their biochemical pattern and later genotyped through the 16S-23S rRNA intergenic spacer PCR amplification and their polymorphism investigation of PCR-amplified 16S-23S rDNA ITS. The main results of the pattern band profile revealed one to four DNA fragments. Distributing 150 E. coli isolates according to their ITS and using RS-PCR, revealed four genotypes and four subtypes. The DNA fragment size ranged from 450 to 550 bp. DNA band patterns analysis revealed considerable genetic diversity in interspecies. Thus, the 450 and 550 bp sizes of the common bands in all E. coli isolates are highly diversified. Genotype I appeared as the most frequent with 77.3% (116 isolates), genotype II with 12% (18 isolates); genotype III with 9.7% (14 isolates), and the IV rarely occurred with 4% (2 isolates). Distributing the E. coli phylogroups showed 84 isolates (56%) of group A, 35 isolates (23.3%) of group B1, 28 isolates (18.7%) of group B2 and only three isolates (2%) of group D.

Comparison of conventional molecular and whole-genome sequencing methods for subtyping Salmonella enterica serovar Enteritidis strains from Tunisia.

We sought to determine the relative value of conventional molecular methods and whole-genome sequencing (WGS) for subtyping Salmonella enterica serovar Enteritidis recovered from 2000 to 2015 in Tunisia and to investigate the genetic diversity of this serotype. A total of 175 Salmonella Enteritidis isolates were recovered from human, animal, and foodborne outbreak samples. Pulsed-field gel electrophoresis (PFGE), multiple locus variable-number tandem repeat analysis (MLVA), and whole-genome sequencing were performed. Eight pulsotypes were detected for all isolates with PFGE (DI = 0.518). Forty-five Salmonella Enteritidis isolates were selected for the MLVA and WGS techniques. Eighteen MLVA profiles were identified and classified into two major clusters (DI = 0.889). Core genome multilocus typing (cgMLST) analysis revealed 16 profiles (DI = 0.785). Whole-genome analysis indicated 660 single-nucleotide polymorphism (SNP) divergences dividing these isolates into 43 haplotypes (DI = 0.997). The phylogenetic tree supported the classification of Salmonella Enteritidis isolates into two distinct lineages subdivided into five clades and seven subclades. Pairwise SNP differences between the isolates ranged between 302 and 350. We observed about 311 SNP differences between the two foodborne outbreaks, while only less or equal to 4 SNP differences within each outbreak. SNP-based WGS typing showed an excellent discriminatory power comparing with the conventional methods such as PFGE and MLVA. Besides, we demonstrate the added value of WGS as a complementary subtyping method to discriminate outbreak from non-outbreak isolates belonging to common subtypes. It is important to continue the survey of Salmonella Enteritidis lineages in Tunisia using WGS.

Nasal colonization and antibiotic resistance patterns of Staphylococcus species isolated from healthy horses in Tripoli, Libya.

The present study investigated the colonization rates and antimicrobial susceptibility of Staphylococcus species isolated from the nostrils of healthy horses. A nonselective laboratory approach was applied, followed by confirmation using a Phoenix automated microbiological system. Among the 92 horses included in the study, 48.9% (45/92) carried Staphylococcus species of mostly the coagulase-negative staphylococci (CoNS) type yielding 70 Staphylococcus strains. Of these strains, 37.1% (26/70; 24 CoNS and 2 coagulase-positive staphylococci; CoPS) were identified as methicillin-resistant staphylococci (MRS) expressing significant resistance to important antimicrobial classes represented mainly by subspecies of CoNS. This is the first study reporting a high prevalence of various Staphylococcus species, particularly strains of CoNS expressing multidrug resistance patterns of public health concern, colonizing healthy horses in Libya.

Dispersal of linezolid-resistant enterococci carrying poxtA or optrA in retail meat and food-producing animals from Tunisia.

OBJECTIVES: The epidemiology of Enterococcus resistant to priority antibiotics including linezolid has mainly been investigated in developed countries and especially in hospitals. We aimed to evaluate the contribution of different non-human reservoirs for the burden of MDR enterococci in Tunisia, where scarce data are available. METHODS: Samples (n = 287) were collected from urban wastewater (n = 57), retail meat (n = 29; poultry/bovine/ovine), milk (n = 89; bovine/ovine), farm animal faeces (n = 80; poultry/bovine/ovine) and pets (n = 32; rabbit/dogs/cats/birds) in different Tunisian regions (2014-17). They were plated onto Slanetz-Bartley agar after pre-enrichment without antibiotics. Standard methods were used for bacterial identification and characterization of antibiotic resistance and virulence genes (PCR), antibiotic susceptibility testing (disc diffusion/broth microdilution; EUCAST/CLSI) and clonality (SmaI-PFGE/MLST). RESULTS: All samples carried Enterococcus (n = 377 isolates) resistant to antibiotics considered to be critical or highly important by WHO. Even without antibiotic selection, 38% of Enterococcus faecalis (Efs) and 22% of Enterococcus faecium (Efm) were identified as MDR. Linezolid-resistant isolates (5%; MIC = 8 mg/L) comprised six poxtA-carrying Efm (cow milk), seven optrA-carrying Efs (chicken faeces/meat) and five Efm lacking cfr/optrA/poxtA (poultry/bovine/ovine/wastewater). Clinically relevant Efm clones (clade A1) were identified in animal/meat sources. Ampicillin resistance (1%) was confined to ST18/ST78-like MDR Efm clones from bovine meat/milk samples carrying relevant virulence markers (e.g. ptsD/IS16). CONCLUSIONS: This study provides evidence of the contribution of livestock and foodstuffs to the dispersal of acquired linezolid resistance genes including poxtA and optrA. We report the first poxtA-carrying Efm in Tunisia, and for the first time in bovine samples, stressing the urgent need for alternative measures to counteract the spread of linezolid-resistant enterococci globally.

Detection of optrA in the African continent (Tunisia) within a mosaic Enterococcus faecalis plasmid from urban wastewaters.

OBJECTIVES: Oxazolidinone resistance is a serious limitation in the treatment of MDR Enterococcus infections. Plasmid-mediated oxazolidinone resistance has been strongly linked to animals where the use of phenicols might co-select resistance to both antibiotic families. Our goal was to assess the diversity of genes conferring phenicol/oxazolidinone resistance among diverse enterococci and to characterize the optrA genetic environment. METHODS: Chloramphenicol-resistant isolates (>16 mg/L, n = 245) from different sources (hospitals/healthy humans/wastewaters/animals) in Portugal, Angola and Tunisia (1996-2016) were selected. Phenicol (eight cat variants, fexA, fexB) or phenicol + oxazolidinone [cfr, cfr(B), optrA] resistance genes were searched for by PCR. Susceptibility (disc diffusion/microdilution), filter mating, stability of antibiotic resistance (500 bacterial generations), plasmid typing (S1-PFGE/hybridization), MLST and WGS (Illumina-HiSeq) were performed for optrA-positive isolates. RESULTS: Resistance to phenicols (n = 181, 74%) and phenicols + oxazolidinones (n = 2, 1%) was associated with the presence of cat(A-8) (40%, predominant in hospitals and swine), cat(A-7) (29%, predominant in poultry and healthy humans), cat(A-9) (2%), fexB (2%) and fexA + optrA (1%). fexA and optrA genes were co-located in a transferable plasmid (pAF379, 72 918 bp) of two ST86 MDR Tunisian Enterococcus faecalis (wastewaters) carrying several putative virulence genes. MICs of chloramphenicol, linezolid and tedizolid were stably maintained at 64, 4 and 1 mg/L, respectively. The chimeric pAF379 comprised relics of genetic elements from different Gram-positive bacteria and origins (human/porcine). CONCLUSIONS: To the best of our knowledge, we report the first detection of optrA in an African country (Tunisia) within a transferable mosaic plasmid of different origins. Its identification in isolates from environmental sources is worrisome and alerts for the need of a concerted global surveillance on the occurrence and spread of optrA.

Staphylococcus aureus isolated from wastewater treatment plants in Tunisia: occurrence of human and animal associated lineages.

The objective was to characterize Staphylococcus aureus isolated from two wastewater treatment plants (WWTPs) located in Tunis City (Tunisia), during the period 2014-2015. Genetic lineages, antibiotic resistance mechanisms and virulence factors were determined for the recovered isolates. S. aureus isolates were recovered from 12 of the 62 wastewater samples tested (19.35%), and one isolate/sample was characterized, all of them being methicillin-susceptible (MSSA). Six spa types (t587, t674, t224, t127, t701 and t1534) were found among the 12 isolates, and the spa-t587, associated with the new sequence type ST3245, was the most predominant one (7 isolates). The remaining isolates were assigned to five clonal complexes (CC5, CC97, CC1, CC6 and CC522) according to the sequence-type determined and/or the spa-type detected. S. aureus isolates were ascribed to agrI (n = 3), agrII (n = 7) and agrIII (n = 1); however, one isolate was non-typeable. S. aureus showed resistance to (number of isolates): penicillin (12), erythromycin (7), tetracycline (one) and clindamycin (one). Among the virulence factors investigated, only one isolate harboured the tst gene, encoding the TSST-1 (toxic shock syndrome toxin 1). Despite the low number of studied isolates, the present study reports the occurrence of both human- and animal-associated S. aureus clonal complexes in WWTPs in Tunisia.

Characterization of Pc promoter variants of class 1 integrons in Escherichia coli isolates from poultry meat.

Integrons are important genetic elements implicated in acquisition and expression of antimicrobial resistance genes. Gene cassettes of class 1 integrons may be differently expressed depending on the Pc promoter variant. Thirty-four Escherichia coli isolates (carrying 38 class 1 integrons), recovered from poultry meat in previous studies in Tunisia and selected by their specific traits, were further characterized in this study. Integron promoter variants and the pulsed-field gel electrophoresis (PFGE) and multilocus sequence typing (MLST) of isolates were determined. Three types of promoter variants were identified among the 38 class 1 integrons (PcW, PcH1, and PcS); the weak promoters were the most predominant. A high clonal diversity of the E. coli strains was demonstrated by PFGE or by MLST. Fifteen PFGE profiles were detected among 19 integron-positive isolates of phylogroup B2, and 12 different sequence types were identified by MLST among the remaining 15 isolates (ST48, ST88, ST101, ST117, ST155, ST189, ST351, ST359, ST410, ST641, ST665, and one new ST). These data reflect that the presence of integrons in these isolates is not due to the clonal dispersion but to dissemination of genetic structures carrying integrons in different clones. To the best of our knowledge, this is the first report examining the gene cassette promoter variants in class 1 integrons of E. coli isolates from poultry meat origin. The predominance of promoters implicated in weak expression/high excision activity of gene cassette arrays is of interest because they could theoretically enhance the capacity of integrons to adapt to antibiotic pressure.

Occurrence of plasmid-mediated quinolone resistance determinants and rmtB gene in Salmonella enterica serovar enteritidis and Typhimurium isolated from food-animal products in Tunisia.

Four hundred and thirty Salmonella isolates, recovered from various food-animal products, were tested for nalidixic acid resistance, plasmid-mediated quinolone resistance, and genetic relationship. One hundred fifteen isolates (113 Salmonella serovar Enteritidis and two Salmonella serovar Typhimurium isolates) of 430 (26.7%) Salmonella isolates exhibited nalidixic acid resistance. Polymerase chain reaction screening for qnrA, qnrB, qnrS, qepA (encoding fluoroquinolones resistance) and rmtB (encoding aminoglycosides resistance) showed that 5 (1.16%) isolates were positive for qnr- and qepA-type genes, and the aac(6')-Ib-cr gene was observed in two (1.7%) Enteritidis isolates concomitantly with qnrA or qnrB. The co-occurrence of qepA and rmtB in one Typhimurium isolate is noteworthy. Pulsed-field gel electrophoresis revealed a high genetic homogeneity of nalidixic-resistant isolates and the persistence of clonal clusters over 4 years in different regions in Tunisia and from various food-animal products. To the best of our knowledge, this is the first report of co-occurrence of qepA and rmtB in a Salmonella strain.

Selection and Characterization of Bacteriocinogenic Lactic Acid Bacteria from the Intestine of Gilthead Seabream (Sparus aurata) and Whiting Fish (Merlangius merlangus): Promising Strains for Aquaculture Probiotic and Food Bio-Preservation.

This study sought to evaluate the probiotic properties and the food preservation ability of lactic acid bacteria isolates collected from the intestines of wild marine fishes (gilthead seabream (Sparus aurata) (n = 60) and whiting fish (Merlangius merlangus) (n = 40)) from the Mediterranean sea in the area of Mostaganem city, Algeria. Forty-two isolates were identified as: Enterococcus durans (n = 19), Enterococcus faecium (n = 15), Enterococcus faecalis (n = 4), Lactococcus lactis subp. lactis (n = 3), and Lactobacillus plantarum (n = 1). All isolates showed inhibition to at least one indicator strain, especially against Listeria monocytogenes, Staphylococcus aureus, Paenibacillus larvae, Vibrio alginolyticus, Enterococcus faecalis, Bacillus cereus, and Bacillus subtilis. In all collected isolates, PCR analysis of enterocin-encoding genes showed the following genes: entP (n = 21), ent1071A/B (n = 11), entB (n = 8), entL50A/B (n = 7), entAS48 (n = 5), and entX (n = 1). Interestingly, 15 isolates harbored more than one ent gene. Antimicrobial susceptibility, phenotypic virulence, and genes encoding virulence factors were investigated by PCR. Resistance to tetracycline (n = 8: tetL + tetK), erythromycin (n = 7: 5 ermA, 2 msrA, and 1 mef(A/E)), ciprofloxacin (n = 1), gentamicin (n = 1: aac(6')-aph(2″)), and linezolid (n = 1) were observed. Three isolates were gelatinase producers and eight were α-hemolytic. Three E. durans and one E. faecium harbored the hyl gene. Eight isolates showing safety properties (susceptible to clinically relevant antibiotics, free of genes encoding virulence factors) were tested to select probiotic candidates. They showed high tolerance to low pH and bile salt, hydrophobicity power, and co-culture ability. The eight isolates showed important phenotypic and genotypic traits enabling them to be promising probiotic candidates or food bio-conservers and starter cultures.

High Biofilm-Forming Ability and Clonal Dissemination among Colistin-Resistant Escherichia coli Isolates Recovered from Cows with Mastitis, Diarrheic Calves, and Chickens with Colibacillosis in Tunisia.

BACKGROUND: Escherichia coli (E. coli) is one of the main etiological agents responsible for bovine mastitis (BM), neonatal calf diarrhea (NCD), and avian colibacillosis (AC). This study aimed to assess resistance and virulence genes content, biofilm-forming ability, phylogenetic groups, and genetic relatedness in E. coli isolates recovered from clinical cases of BM, NCD, and AC. MATERIALS/METHODS: A total of 120 samples including samples of milk (n = 70) and feces (n = 50) from cows with BM and calves with NCD, respectively, were collected from different farms in Northern Tunisia. Bacterial isolation and identification were performed. Then, E. coli isolates were examined by disk diffusion and broth microdilution method for their antimicrobial susceptibility and biofilm-forming ability. PCR was used to detect antimicrobial resistance genes (ARGs), virulence genes (VGs), phylogenetic groups, and Enterobacterial repetitive intergenic consensus PCR (ERIC-PCR) for their clonal relationship. RESULTS: Among the 120 samples, 67 E. coli isolates (25 from BM, 22 from AC, and 20 from NCD) were collected. Overall, 83.6% of isolates were multidrug resistant. Thirty-six (53.73%) isolates were phenotypically colistin-resistant (CREC), 28.3% (19/67) were ESBL producers (ESBL-EC), and forty-nine (73.1%) formed biofilm. The blaTEM gene was found in 73.7% (14/19) of isolates from the three diseases, whilst the blaCTXM-g-1 gene was detected in 47.3% (9/19) of isolates, all from AC. The most common VG was the fimA gene (26/36, 72.2%), followed by aer (12/36, 33.3%), cnf1 (6/36, 16.6%), papC (4/36, 11.1%), and stx1 and stx2 genes (2/36; 5.5% for each). Phylogenetic analysis showed that isolates belonged to three groups: A (20/36; 55.5%), B2 (7/36; 19.4%), and D (6/36; 16.6%). Molecular typing by ERIC-PCR showed high genetic diversity of CREC and ESBL E. coli isolates from the three animal diseases and gave evidence of their clonal dissemination within farms in Tunisia. CONCLUSION: The present study sheds new light on the biofilm-forming ability and clonality within CREC and ESBL-EC isolated from three different animal diseases in Tunisian farm animals.

Occurrence of High-Risk Clonal Lineages ST58, ST69, ST224, and ST410 among Extended-Spectrum ß-Lactamase-Producing Escherichia coli Isolated from Healthy Free-Range Chickens (Gallus gallus domesticus) in a Rural Region in Tunisia.

Antimicrobial-resistant Escherichia coli isolates have emerged in various ecologic compartments and evolved to spread globally. We sought to (1.) investigate the occurrence of ESBL-producing E. coli (ESBL-Ec) in feces from free-range chickens in a rural region and (2.) characterize the genetic background of antimicrobial resistance and the genetic relatedness of collected isolates. Ninety-five feces swabs from free-range chickens associated with two households (House 1/House 2) in a rural region in northern Tunisia were collected. Samples were screened to recover ESBL-Ec, and collected isolates were characterized for phenotype/genotype of antimicrobial resistance, integrons, and molecular typing (pulsed-field gel electrophoresis (PFGE) and multilocus sequence typing (MLST)). Overall, 47 ESBL-Ec were identified, with the following genes detected: 35 blaCTX-M-1, 5 blaCTX-M-55, 5 blaCTX-M-15, 1 blaSHV-2, and 1 blaSHV-12. Resistance to fluoroquinolones, tetracycline, sulfonamides, and colistin was encoded by aac(6')-Ib-cr (n = 21), qnrB (n = 1), and qnrS (n = 2); tetA (n = 17)/tetB (n = 26); sul1 (n = 29)/sul2 (n = 18); and mcr-2 (n = 2) genes, respectively. PFGE and MLST identified genetic homogeneity of isolates in House 1; however, isolates from House 2 were heterogeneous. Notably, among nine identified sequence types, ST58, ST69, ST224, and ST410 belong to pandemic high-risk clonal lineages associated with extrapathogenic E. coli. Minor clones belonging to ST410 and ST471 were shared by chickens from both households. The virulence genes fyuA, fimH, papGIII, and iutA were detected in 35, 47, 17, and 23 isolates, respectively. Findings indicate a high occurrence of ESBL-Ec in free-range chickens and highlight the occurrence of pandemic zoonotic clones.