Prevalence, virulence genes, and antimicrobial profiles of Escherichia coli O157:H7 isolated from healthy cattle in Tunisia.

INTRODUCTION: Shiga toxin-producing Escherichia coli (STEC) O157:H7 is associated with intestinal infection in humans and is considered an important cause of food-borne diseases. The aim of the study was to assess the incidence of E. coli O157:H7 in fecal samples of healthy cattle collected in slaughterhouses (n = 160) and from five farms (n = 100). METHODOLOGY: E. coli isolates were detected on MacConkey agar. A total of 236 E. coli isolates were recovered from fecal samples of healthy cattle. We used sorbitol MacConkey medium to detect non-sorbitol fermenting colonies. These bacteria were examined for the presence of O157:H7 antigen by latex agglutination. The isolation of E. coli O157:H7 has been confirmed with PCR amplification of rfbEO157 and fliCH7 specific genes for serogroup O157 and with multiplex PCR of stx1, stx2, eaeA, and ehxA. All isolates were examined for their susceptibility to 21 antibiotics by the disc diffusion method. RESULTS: Of the 236 E. coli isolates, 4.2% (10/236) were positive for STEC O157:H7. Shiga toxin gene (stx2) and ehxA were present in 70% of isolates, stx1 and eae were confirmed in 60% of the isolates. Other virulence factors screened (fimH, sfa/focDE, cdt3, traT, iutA, and hlyA) were present among the 10 isolates. All E. coli O157:H7 isolates were sensitive to amoxicillin/clavulanic acid, cefotaxime, cefepime, aztreonam, colistin, and sulfamethoxazole/trimethoprim. All isolates belong to the phylo-group E. CONCLUSIONS: This is the first study of the incidence of E. coli O157:H7 in cattle in Tunisia. Our finding proves the existence of STEC O157:H7 in healthy animals producing food for human consumption which could be a source of food-borne disease.

Prevalence, risk factors and emergence of extended-spectrum ß-lactamase producing-, carbapenem- and colistin-resistant Enterobacterales isolated from wild boar (Sus scrofa) in Tunisia.

Antimicrobial resistance (AMR) is recognized as an emerging and growing public health problem worldwide. In Tunisia, knowledge is still limited to domestic animals and humans, and only few data are available regarding the role of wildlife. This research determined the antibiotic susceptibility profiles of Beta-lactamase producing Gram-negative bacteria isolated from the faeces of 110 wild boars (Sus scrofa) in northern Tunisia. Fecal samples, obtained post mortem from boar carcasses, were cultured on MacConkey agar and MacConkey agar containing 2 mg/L of cefotaxime. A total of 102 Enterobacterales isolates were identified from 94(85%) fecal samples. Escherichia coli (56, 54%), Citrobacter freundii (14, 13%), Klebsiella oxytoca (11, 10%), and Klebsiella pneumoniae (7, 6%) were the most predominantly identified Enterobacterales. However, Pantoea spp. (4, 4%), Enterobacter spp. (3,3%), Enterobacter cloacae (1, 1%), Enterobacter gergoviae (2, 2%), Proteus mirabilis (2, 2%), Yersinia sp. (1, 1%), and Citrobacter diversus (1, 1%) were rarely identified. Antimicrobial susceptibility tests revealed that 55% (57/102) of the identified strains were multidrug resistant (MDR). A total of 30% (31/102) of the tested isolates were recognized as Extended Spectrum ß-Lactamase (ESBL)-producing strains and blaCTX-M-G1, blaTEM, blaSHV ß-lactamases were the main encoding genes revealed. Furthermore, identified isolates showed a high level of AMR, especially for amoxicillin-clavulanic acid (77.67%), ticarcillin-clavulanic acid (71.85%), streptomycin (76.69%), amoxicillin (75.73%), and cephalotin (74.76%). Alarming levels of resistance to colistin (2.9%) and ertapenem (9.7%) were revealed and confirmed by the detection of mcr-1, and blaIMP and blaVIM genes, respectively. Various phenotypes of AMR were obtained in this study highlighting the important role of wild boars as hosts and even carriers for several resistant Enterobacterales isolates. This may represents a focal risk factor allowing the transmission of these strains between domestic, wild animals, environment and humans.

Prevalence of Escherichia coli O157:H7 isolated from fecal samples of diarrheic camels in Tunisia.

Shiga­toxin­producing E. coli (STEC) is a foodborne pathogen associated with outbreaks worldwide that can be identified in the feces and in the meat of food­producing animals. Our study aimed to evaluate the incidence of E. coli O157:H7 in the feces of diarrheic camels (Camelus dromedarius) in Tunisia. From January 2018 to April 2019, 120 unduplicated fecal samples were obtained from diarrheic camels located in southern Tunisia. Non­sorbitol­fermenting colonies were confirmed as E. coli O157 via latex agglutination test and were screened for the presence of rfbEO157, fliCH7, stx1, stx2, eaeA, and ehxA genes by PCR. All isolates were examined for their susceptibility to 21 antibiotics. Of the 70 E. coli isolates that were recovered from 120 diarrheic camels, 4 (5.7%) were identified as STEC O157:H7. All isolates harbored ehxA and eae genes. Shiga toxin genes stx2 and stx1 were present in 50% and 25% of isolates, respectively. All E. coli O157:H7 isolates were sensitive to amoxicillin/clavulanic acid, cefotaxime, cefepime, aztreonam, colistin, and sulfamethoxazole­trimethoprim. All isolates belonged to the phylogroup E. This is the first report of E. coli O157:H7 isolates from diarrheic camels in Tunisia with a prevalence of 4 isolates (3.3%) amongst 120 fecal samples. This study supports the necessity for a platform purposed for regular screening and surveillance programs in food­producing animals and meat products, to perform early and rapid identification of food­borne pathogens.

First Report of Antimicrobial Susceptibility and Virulence Gene Characterization Associated with Staphylococcus aureus Carriage in Healthy Camels from Tunisia.

A total of 318 nasal and rectal swabs were collected from 159 apparently healthy camels (Camelus dromedarius) randomly selected from five regions in southern and central Tunisia and screened for Staphylococcus aureus carriage. Staphylococcus spp. were recovered from 152 of 159 camels studied (95.6%) and in total 258 swabs (81%) were positive. Among these isolates, 16 were coagulase positive Staphylococcus (CoPS) (6.2%) and were characterized by biochemical and molecular tests as S. aureus. These were isolated from 14 camels (8.8%) with co-carriage in nasal and rectal mucosa by two camels. All S. aureus isolates recovered were methicillin-susceptible Staphylococcus aureus (MSSA) and were characterized by spa typing and PFGE. Three different spa types were recovered: t729, t4013 and a spa type newly registered as t19687, which was the most common. PFGE analysis revealed seven different patterns and these were characterized by MLST, which revealed five different sequence types (ST6, ST88, ST3583 and two new sequences, ST6504 and ST6506). All isolates harbored different virulence genes, including hld, encoding delta hemolysin; lukE-lukD, encoding bicomponent leukotoxin LukE-LukD; the clfB gene, encoding clumping factor B; the laminin gene, encoding laminin-binding protein; and cap8, encoding capsule type 8. Fifteen isolates harbored hemolysin beta (hlb) and fourteen encoded hemolysin alpha (hla) and hemolysin G2 (hlgv). Adhesin factors, including clfA and fnbB, were detected in five and four isolates respectively. Binding proteins, including collagen (cbp) and elastin-binding protein (ebp), were detected in two S. aureus isolates while fibrinogen-binding protein (fib) was identified in four isolates. This study provides the first set of genotyping data on the population structure and presence of toxin genes of S. aureus strains in Tunisian camels.

Multiple Introductions of Rabbit Hemorrhagic Disease Virus Lagovirus europaeus/GI.2 in Africa.

Rabbit hemorrhagic disease (RHD) causes high mortality and morbidity in European rabbits (Oryctolagus cuniculus). In Africa, the presence of the causative agent, the rabbit hemorrhagic disease virus (RHDV), was first confirmed in 1992 (genotype Lagovirus europaeus/GI.1). In 2015, the new genotype Lagovirus europaeus/GI.2 (RHDV2/b) was detected in Tunisia. Currently, GI.2 strains are present in several North and Sub-Saharan African countries. Considerable economic losses have been observed in industrial and traditional African rabbitries due to RHDV. Like other RNA viruses, this virus presents high recombination rates, with the emergence of GI.2 being associated with a recombinant strain. Recombination events have been detected with both pathogenic (GI.1b and GII.1) and benign (GI.3 and GI.4) strains. We obtained complete genome sequences of Tunisian GI.2 strains collected between 2018 and 2020 and carried out phylogenetic analyses. The results revealed that Tunisian strains are GI.3P-GI.2 strains that were most likely introduced from Europe. In addition, the results support the occurrence of multiple introductions of GI.2 into Africa, stressing the need for characterizing complete genome sequences of the circulating lagoviruses to uncover their origin. Continued monitoring and control of rabbit trade will grant a better containment of the disease and reduce the disease-associated economic losses.

First serological evidence of the Rift Valley fever Phlebovirus in Tunisian camels.

Rift Valley fever (RVF) is a mosquito-borne zoonosis that severely impacts livelihoods, national and international economies, and human health. Few studies have investigated the prevalence of this infection in Tunisian livestock. The present report aimed to update the epidemiological status and identify the risk factors associated with this RVF virus infection in the one-humped dromedary camel from arid areas. A total of 470 sera of apparently healthy camels (Camelus dromedarius) were collected from six governorates from southern and central Tunisia. Samples were tested by a competitive Enzyme Linked Immunosorbent Assay (ELISA). An overall, 162 camels (34%, 95%CI: 0.1-0.4) were seropositive to RVF virus antigen. Logistic regression model revealed three potential risk factors associated with the infection. A meaningful high seropositivity was observed among aged camels (>10 years-old) (40%) (P=0.001; OR=3.367). Besides, camels raised in small flocks particularly intended for meat production showed a high level of seropositivity (37%) (P=0.013; OR=13.173). Animals having close contact with other ruminants showed high seroprevalence (37%) (P=0.022; OR=10.919). This report indicated that Tunisian one-humped dromedaries were exposed to this virus and may contribute to its dissemination among farmers and other livestock. Furthers studies are urgently required to isolate and characterize this virus, evaluate the potential risk of human infection particularly in farmers, veterinarians and slaughterhouse workers and finally to program a serious strategy for RVF control.

High Genetic Diversity of Enterobacteriaceae Clones and Plasmids Disseminating Resistance to Extended-Spectrum Cephalosporins and Colistin in Healthy Chicken in Tunisia.

Enterobacteriaceae resistant to extended-spectrum cephalosporins (ESC-R) are listed as "priority pathogens" by the World Health Organization, and the Agri-food sector has regularly been pointed out as a potential source of ESC-R for humans through food consumption and animal handling. Chicken industry and chicken meat have recurrently been under specific scrutiny due to the high proportions of ESC-R reported worldwide in this sector. In Tunisia, recent studies suggested that the plasmidic AmpC blaCMY-2 gene may have emerged in chicken. We thus collected 258 cloacal swabs from five different farms and selected ESC-R isolates to determine the current ESC-R prevalence and epidemiology. All five farms were ESC-R positive with proportions ranging from 4% to 67.3%. blaCTX-M-1/IncI1/ST3 was the dominant gene/plasmid association in chicken, but several other CTX-M genes and plasmid backgrounds were shown to spread ESC-R. Surprisingly, the CMY-2 enzyme was only identified in one isolate. In addition, we also reported the sporadic presence of the mcr-1 gene carried by an IncHI2 plasmid. Our data suggest that the high diversity of Enterobacteriaceae clones and plasmids circulating in healthy chicken in Tunisia maintains a high ESC-R proportion in flocks and constitutes a major source of ESC-R determinants further disseminating in the food chain.

Various Inc-type plasmids and lineages of Escherichia coli and Klebsiella pneumoniae spreading blaCTX-M-15,blaCTX-M-1 and mcr-1 genes in camels in Tunisia.

OBJECTIVES: Resistance to extended-spectrum cephalosporins, fluoroquinolones and colistin is under constant scrutiny in food-producing animals worldwide. However, little is known about camels, which provide milk and meat for human consumption, and are attractions for tourists to ride in arid regions. This study assessed the role of camels as potential reservoirs of these resistance determinants. METHODS: Faecal swabs were collected from 232 camels in Tunisia between April 2016 and July 2018. Enterobacteriaceae were detected on MacConkey agar and extended-spectrum ß-lactamase (ESBL)-producers on the same medium supplemented with cefotaxime. Antimicrobial resistance was assessed by disc diffusion, and ESBL-producing isolates were further characterised by phylogrouping (for Escherichia coli, E. coli) and multilocus sequence typing. Genetic support of the blaESBL and mcr-1 genes was identified by plasmid-typing and Southern blot. RESULTS: E. coli were identified in 163 of 232 (70.3%) and Klebsiella pneumoniae (K. pneumoniae) in 16 of 232 (6.9%) of the dominant flora. Three E. coli and one K. pneumoniae (1.3% and 0.4%, respectively) were found on cefotaxime-enriched media. One K. pneumoniae and one E. coli from a tourist farm harboured the blaCTX-M-15 gene on an IncY plasmid, while the two E. coli from the butchery sector displayed the blaCTX-M-15 gene on an IncI1 plasmid and colocalisation of the blaCTX-M-1 and mcr-1 genes on an IncHI2 plasmid. CONCLUSIONS: This study reported ESBL-producing Enterobacteriaceae in Tunisian camels from both tourist and meat-producing sectors. This was the first description of the mcr-1 gene in a meat-producing camel. Although not alarming, this context needs specific attention to avoid camels becoming a bigger reservoir for multidrug-resistant Enterobacteriaceae.

Epidemiology, Antimicrobial Resistance, and Extended-Spectrum Beta-Lactamase-Producing Enterobacteriaceae in Clinical Bovine Mastitis in Tunisia.

Bovine mastitis is a major disease in dairy cattle that causes high economic losses annually. Staphylococci, streptococci, and coliforms are among the major pathogens responsible for such infections. While data on bovine mastitis are numerous in Europe where the efficacy of farm management was monitored, those are scarce in African countries. In this study, we reported the occurrence of Escherichia coli (118/372, 31.7%) and Klebsiella pneumoniae (77/372, 20.7%), two environmental pathogens known to cause bovine mastitis. Resistance phenotypes were frequently identified for tetracycline (E. coli, 46.6%/K. pneumoniae, 20.8%), sulfonamides-trimethoprim (17.8%/11.7%), gentamicin (19.5%/14.3%), and enrofloxacin (11.0%/6.5%). No carbapenem-resistant isolate was detected. Extended-spectrum beta-lactamases (ESBLs) were detected on selective medium in three E. coli and six K. pneumoniae, all carrying the blaCTX-M-15 gene. The K. pneumoniae belonged to two highly uncommon sequence types (ST471 and ST1083), while E. coli clustered in the ST167/617 clones, which have been widely reported in humans, animals, and the environment. These data point out the necessity to improve farm management in Tunisia to reduce the occurrence of coliform-induced mastitis and to avoid the dissemination in this sector of ESBL-producing E. coli and K. pneumoniae, which are of public health concern.