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.

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.

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.

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.

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.

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 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.

Hiding in plain sight-wildlife as a neglected reservoir and pathway for the spread of antimicrobial resistance: a narrative review.

Antimicrobial resistance represents a global health problem, with infections due to pathogenic antimicrobial resistant bacteria (ARB) predicted to be the most frequent cause of human mortality by 2050. The phenomenon of antimicrobial resistance has spread to and across all ecological niches, and particularly in livestock used for food production with antimicrobials consumed in high volumes. Similarly, hospitals and other healthcare facilities are recognized as significant 'hotspots' of ARB and antimicrobial resistance genes (ARGs); however, over the past decade, new and previously overlooked ecological niches are emerging as hidden reservoirs of ARB/ARGs. Increasingly extensive and intensive industrial activities, degradation of natural environments, burgeoning food requirements, urbanization, and global climatic change have all dramatically affected the evolution and proliferation of ARB/ARGs, which now stand at extremely concerning ecological levels. While antimicrobial resistant bacteria and genes as they originate and emanate from livestock and human hosts have been extensively studied over the past 30 years, numerous ecological niches have received considerably less attention. In the current descriptive review, the authors have sought to highlight the importance of wildlife as sources/reservoirs, pathways and receptors of ARB/ARGs in the environment, thus paving the way for future primary research in these areas.

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.

Genetic characterization of ESBL/pAmpC-producing Escherichia coli isolated from forest, urban park and cereal culture soils.

Little is known about the role of forestland and non-fertilized agriculture soils as reservoirs of extended-spectrum beta-lactamase (ESBL) and plasmid-borne AmpC (pAmpC)-producing Escherichia coli isolates. Thus, in the present study, 210 soil samples from various origins (forest of Oued Zen (Ain Drahem), non-agriculture soils from different park gardens in Tunis City, cereal culture soils and home gardens) were investigated to characterize cefotaxime-resistant E. coli isolates. A total of 22 ESBL/pAmpC-producing E. coli were collected, and all harbored variants of the blaCTX-M gene (15 blaCTX-M-1, 5 blaCTX-M-55 and 2 blaCTX-M-15). A total of seven and two isolates harbored also blaEBC and blaDHA-like genes, respectively. Resistances to tetracycline, sulfonamides and fluoroquinolones were encoded by tetA (n = 4)/tetB (n = 12), sul1 (n = 17)/sul2 (n = 19) and aac(6')-Ib-cr (n = 2)/qnrA (n = 1)/qnrS (n = 1) genes, respectively. A total of seven isolates were able to transfer by conjugation cefotaxime-resistance in association or not with other resistance markers. PFGE showed that ten and two isolates were clonally related (pulsotypes P1 and P2). The 10 P1 isolates had been collected from forestland, cereal culture soils and an urban park garden in Tunis City, arguing for a large spread of clonal strains. Our findings highlight the occurrence of ESBL/pAmpC-E. coli isolates in soils under limited anthropogenic activities and the predominance of CTX-M enzymes that are largely disseminated in E. coli from humans and animals in Tunisia.

Genetic Background of Antimicrobial Resistance in Multiantimicrobial-Resistant Escherichia coli Isolates from Feces of Healthy Broiler Chickens in Tunisia.

Multiantimicrobial-resistant Escherichia coli isolates are a global human health problem causing increasing morbidity and mortality. Genes encoding antimicrobial resistance are mainly harbored on mobile genetic elements (MGEs) such as transposons and plasmids as well as integrons, which enhance their rapid spread. The aim of this study was to characterize 83 multiantimicrobial-resistant E. coli isolates recovered from healthy broiler chickens. Among 78 tetracycline-resistant isolates, the tetA, tetB, and tetC genes were detected in 59 (75.6%), 14 (17.9%), and one (1.2%) isolates, respectively. The sul1, sul2, and sul3 genes were detected 31 (46.2%), 16 (23.8%), and 6 (8.9%) isolates, respectively, among 67 sulfonamide-resistant isolates. The PCR-based replicon typing method showed plasmids in 29 isolates, IncFIB (19), IncI1-Iγ (17), IncF (14), IncK (14), IncFIC (10), IncP (8), IncY (3), IncHI2 (1), and IncX (1). The class 1 and 2 integrons were detected in 57 and 2 isolates, respectively; one isolate harbored both integrons. Seven and one gene cassette arrays were identified in class 1 and class 2 integrons, respectively. Our findings show that multiantimicrobial-resistant E. coli isolates from chickens serve as reservoirs of highly diverse and abundant tet and sul genes and plasmid replicons. Such isolates and MGEs pose a potential health threat to the public and animal farming.

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.

Prevalence and antimicrobial resistance of Staphylococcus species isolated from cats and dogs.

Background: Methicillin-resistant staphylococci (MRS) are an emerging global problem with serious public health concern. Aims: This study investigated the prevalence and antimicrobial susceptibility of commensal Staphylococcus species isolated from healthy and clinical cats and dogs. Methods: Nasal swab samples were collected from animals and processed using selective and semi-selective mediums. Presumptive isolates were subjected to biochemical testing and analyzed using the Phoenix automated identification and susceptibility testing system. PCRs protocols were used to screen for mecA and pvl genes. Results: In total, 151 pets (103 cats and 48 dogs) were enrolled, of which 14 dogs (29%) and 24 cats (23%) were colonized with various Staphylococcus species mainly originated from healthy animals. A total of 38 staphylococci isolates were collected and distributed between 24 coagulase-negative and 14 coagulase-positive staphylococci. Only 13 staphylococci strains were identified as MRS, out of which only five isolates expressed that the mecA gene exclusively originated from healthy pets. Conclusion: This is the first study reporting the prevalence and colonization status of staphylococci species and MRS strains isolated from cats and dogs in Libya. The study reports important information of medical and clinical importance on antimicrobial and multidrug resistance of different staphylococci strains, particularly the coagulase negative species.

High Frequency and Diversity of Tetracycline Resistance Genes in the Microbiota of Broiler Chickens in Tunisia.

Tetracycline resistance is still considered one of the most abundant antibiotic resistances among pathogenic and commensal microorganisms. The aim of this study was to evaluate the prevalence of tetracycline resistance (tet) genes in broiler chickens in Tunisia, and this was done by PCR. Individual cloacal swabs from 195 broiler chickens were collected at two slaughterhouses in the governorate of Ben Arous (Grand Tunis, Tunisia). Chickens were from 7 farms and belonged to 13 lots consisting of 15 animals randomly selected. DNA was extracted and tested for 14 tet genes. All the lots examined were positive for at least 9 tet genes, with an average number of 11 tet genes per lot. Of the 195 animals tested, 194 (99%) were positive for one or more tet genes. Tet(L), tet(M) and tet(O) genes were found in 98% of the samples, followed by tet(A) in 90.2%, tet(K) in 88.7% and tet(Q) in 80%. These results confirm the antimicrobial resistance impact in the Tunisian poultry sector and suggest the urgent need to establish a robust national antimicrobial resistance monitoring plan. Furthermore, the molecular detection of antibiotic resistance genes directly in biological samples seems to be a useful means for epidemiological investigations of the spread of resistance determinants.

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.

Genetic characterization of extended-spectrum ß-lactamase-producing Enterobacteriaceae from a biological industrial wastewater treatment plant in Tunisia with detection of the colistin-resistance mcr-1 gene.

This study evaluated the occurrence of extended-spectrum ß-lactamases (ESBL) and associated resistance genes, integrons, and plasmid types, as well as the genetic relatedness of enterobacterial isolates in the wastewater treatment plant (WWTP) of La Charguia, Tunis City (Tunisia). A total of 100 water samples were collected at different points of the sewage treatment process during 2017-2019. Antimicrobial susceptibility was conducted by the disc-diffusion method. blaCTX-M, blaTEM and blaSHV genes as well as those encoding non-ß-lactam resistance, the plasmid types, occurrence of class1 integrons and phylogenetic groups of Escherichia coli isolates were determined by PCR/sequencing. Genomic relatedness was determined by multi-locus sequence typing (MLST) for selected isolates. In total, 57 ESBL-producer isolates were recovered (47 E. coli, eight Klebsiella pneumoniae, 1 of the Citrobacter freundii complex and 1 of the Enterobacter cloacae complex). The CTX-M-15 enzyme was the most frequently detected ESBL, followed by CTX-M-27, CTX-M-55 and SHV-12. One E. coli isolate harboured the mcr-1 gene. The following phylogroups/sequence types (STs) were identified among ESBL-producing E. coli isolates: B2/ST131 (subclade-C1), A/ST3221, A/ST8900, D/ST69, D/ST2142, D/ST38, B1/ST2460 and B1/ST6448. High numbers of isolates harboured the class 1 integrons with various gene cassette arrays as well as IncP-1 and IncFIB plasmids. Our findings confirm the importance of WWTPs as hotspot collectors of ESBL-producing Enterobacteriaceae with a high likelihood of spread to human and natural environments.

Genetic characterization of ESBL-producing Escherichia coli and Klebsiella pneumoniae isolated from wastewater and river water in Tunisia: predominance of CTX-M-15 and high genetic diversity.

Aquatic environments are crucial hotspots for the dissemination of antibiotic resistant microorganisms and resistance genes. Thus, the purpose of this study was to investigate the occurrence and the genetic characterization of cefotaxime-resistant (CTXR) Enterobacteriaceae at a Tunisian semi-industrial pilot plant with biological treatment (WWPP) and its receiving river (Rouriche River, downstream from WWPP) located in Tunis City, during 2017-2018. We collected 105 and 15 water samples from the WWPP and the Rouriche River, respectively. Samples were screened to recover ESBL-producing Enterobacteriaceae (ESBL-E) and isolates were characterized for phenotype/genotype of antimicrobial resistance, integrons, plasmid types and molecular typing (multilocus sequence typing, MLST). Among 120 water samples, 33 and 4 contained ESBL-producing E. coli and K. pneumoniae isolates, respectively. Most isolates were multidrug resistant and produced CTX-M-15 (28 isolates), CTX-M-1 (4 isolates), CTX-M-55 (2 isolates), CTX-M-27 (one isolate), SHV-12 (one isolate) and VEB beta-lactamases (one isolate). All K. pneumoniae were CTX-M-15-positive. Four colistin-resistant isolates were found (MIC 4-8 µg/ml), but they were negative for the mcr genes tested. Class 1 integrons were detected in 21/25 trimethoprim/sulfamethoxazole-resistant isolates, and nine of them carried the gene cassette arrays: aadA2 + dfrA12 (n = 4), aadA1 + dfrA15 (n = 2), aadA5 + dfrA17 (n = 2) and aadA1/2 (n = 1). The IncP and IncFIB plasmids were found in 30 and 16 isolates, respectively. Genetic lineages detected were as follows: E. coli (ST48-ST10 Cplx, ST2499, ST906, ST2973 and ST2142); K. pneumoniae: (ST1540 and ST661). Our findings show a high rate of CTX-M-15 and high genetic diversity of ESBL-E isolates from WWPP and receiving river water.