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

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

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

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

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

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

Beyond the Wild MRSA: Genetic Features and Phylogenomic Review of mecC-Mediated Methicillin Resistance in Non-aureus Staphylococci and Mammaliicocci.

Methicillin resistance, mediated by the mecA gene in staphylococci and mammaliicocci, has caused tremendous setbacks in the use of antibiotics in human and veterinary medicine due to its high potential of presenting the multidrug resistance (MDR) phenotype. Three other mec analogs exist, of which the mecC has evolutionary been associated with methicillin-resistant Staphylococcus aureus (MRSA) in wild animals, thus loosely referred to as the wild MRSA. In this study, we present an epidemiological review and genomic analysis of non-aureus staphylococci and mammaliicocci that carry the mecC-mediated methicillin resistance trait and determine whether this trait has any relevant link with the One Health niches. All previous studies (2007 till 2023) that described the mecC gene in non-aureus staphylococci and mammaliicocci were obtained from bibliometric databases, reviewed, and systematically analyzed to obtain the antimicrobial resistance (AMR) and virulence determinants, mobilome, and other genetic contents. Moreover, core genome single-nucleotide polymorphism analysis was used to assess the relatedness of these strains. Of the 533 articles analyzed, only 16 studies (on livestock, environmental samples, milk bulk tanks, and wild animals) were eligible for inclusion, of which 17 genomes from 6 studies were used for various in silico genetic analyses. Findings from this systematic review show that all mecC-carrying non-aureus staphylococci were resistant to only beta-lactam antibiotics and associated with the classical SCCmec XI of S. aureusLGA251. Similarly, two studies on wild animals reported mecC-carrying Mammaliicoccus stepanovicii associated with SCCmec XI. Nevertheless, most of the mecC-carrying Mammaliicoccus species presented an MDR phenotype (including linezolid) and carried the SCCmec-mecC hybrid associated with mecA. The phylogenetic analysis of the 17 genomes revealed close relatedness (<20 SNPs) and potential transmission of M. sciuri and M. lentus strains in livestock farms in Algeria, Tunisia, and Brazil. Furthermore, closely related M. sciuri strains from Austria, Brazil, and Tunisia (<40 SNPs) were identified. This systematic review enhances our comprehension of the epidemiology and genetic organization of mecC within the non-aureus staphylococci and mammaliicocci. It could be hypothesized that the mecC-carrying non-aureus staphylococci are evolutionarily related to the wild MRSA-mecC. The potential implications of clonal development of a lineage of mecA/mecC carrying strains across multiple dairy farms in a vast geographical region with the dissemination of MDR phenotype is envisaged. It was observed that most mecC-carrying non-aureus staphylococci and mammaliicocci were reported in mastitis cases. Therefore, veterinarians and veterinary microbiology laboratories must remain vigilant regarding the potential existence of mecA/mecC strains originating from mastitis as a potential niche for this resistance trait.

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

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