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.

Physicochemical and Microbiological Water Quality Assessment of a Northwestern Algerian Dam: Detection of Ichtyopathogenic Bacteria.

Freshwater fish are often exposed to threats from anthropogenic or natural origins, such as pathogenic or opportunistic microorganisms responsible for a broad range of severe infections. In this study, we aimed to assess this microbiological threat to fish in an Algerian northwestern dam Sekkak (Tlemcen) by evaluating the diversity of ichtyopathogenic bacteria. In order to determine the water quality, physicochemical analyses of the dam water were carried out in situ. Ichtyopathogenic bacteria were isolated on selective media and identified by API galleries and molecular techniques (PCR and sequencing of the 16S rRNA gene). Besides, the antibiograms were constructed for all the isolates. The physicochemical and bacteriological analyses allowed us to classify the dam water as moderately polluted to polluted. Furthermore, an important diversity of ichtyopathogenic bacterial species was observed as Aeromonas hydrophila, Providencia rettgeri, and Pseudomonas aeruginosa were retrieved. The antibiogram test revealed notable resistance. The antibiotic family for which most resistances were found was the ß-lactam family, followed by aminoglycosides and macrolides. These results indicate that aquatic environments can shelter multidrug-resistant pathogenic bacteria representing a threat to the endemic fauna. Therefore, it is important to closely monitor these waters in order to improve the fish's living environment and ensure healthier production.

Reproductive Performance of Tunisian Arabian Stallions: A Study on the Variance and Estimation of Heritability.

A consistently high level of stallion fertility plays an economically important role in modern horse breeding. To better understand the factors affecting variation in stallion fertility, we have performed a statistical analysis study on some breeding factors: year of breeding, stud farm, age of the stallion, number of covered mares per stallion, reproduction methods, and age of the mare. This work was conducted on 94 purebred Arabian stallions in four different regions of Tunisia. The results showed an increase in the number of stallions during the study period, ranging from 11.33% in 2011 to 13.29% in 2018. Sidi Thabet's stud farm contained the highest number of purebred Arabian stallions. The majority of stallions were between 15 and 21 years old and had covered 1 to 20 mares; 95.19% of stallions were used in natural mating (Nat); 50.36% had low fertility, 17.69% had medium fertility, and 32.3% had excellent fertility according to fertility standards. Depending on the year and stud, there was a variation in fertility per cycle (FERPCE) and end-of-season fertility (FERPSE) of the stallions. The highest average FERPCE and FERPSE values were obtained using artificial insemination with fresh semen (AIF). Analysis of FERPCE and FERPSE showed that the model used in our study explained 40.21% of total variability observations for FERPCE and 42.1% for FERPSE. The used statistical model showed that the breeding year, the stud, the age of the stallion, the number of covered mares by stallions and the method of reproduction significantly affected both FERPCE and FERPSE (with p = 0.001). Low to moderate heritability estimations for FERPCE (hs2 = 0.08) and FERPSE (hes2 = 0.36) were obtained.

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.

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.

Molecular and immunohistochemical analysis of BRAF gene in primary cutaneous melanoma: Discovery of novel mutations.

BACKGROUND: Determination of BRAF status is mandatory for targeted therapy but it is not currently available in most developing countries. AIM: We aimed to analyze BRAF mutations in a series of cutaneous melanoma of Tunisian patients and to compare BRAF V600E mutation detection by immunohistochemistry to DNA sequencing. PATIENTS AND METHODS: Archival formalin fixed paraffin embedded tissues from 28 patients with primary cutaneous melanoma were evaluated for the BRAF mutations by Sanger sequencing and immunohistochemistry. RESULTS: BRAF mutations were detected in 19/28 (68%) of analyzed tumors. The sensitivity and specificity of immunohistochemistry compared to DNA sequencing for identification of the BRAF V600E mutation were 100%. We found five novel mutations, one of them had deleterious effect. CONCLUSION: The present study shows an intermediate frequency of mutations of the BRAF gene in cutaneous melanoma of Tunisian patients, and supports the use of immunohistochemistry as a screening test for the assessment of the BRAF V600E status in the management of melanoma in clinical practice.

First Report of NDM and VIM Coproducing Klebsiella pneumoniae in Tunisia and Emergence of Novel Clones.

Metallo-ß-lactamase (MBL) producing bacteria constitute nowadays a serious global concern worldwide. The purpose of our present study was to characterize molecular features of MBL producing bacteria and to identify the existing clones in our area. Thirteen MBL-producing-Klebsiella pneumoniae were detected in clinical samples from patients hospitalized in the Military hospital of Tunisia during 2017. The molecular research by polymerase chain reaction and sequencing of gene encoding MBL enzymes showed that only two types were identified in our study: blaNDM-1 and blaVIM-1 genes detected, respectively, in eight and six isolates. An association between these two MBL genes (blaNDM-1+blaVIM-1) has been observed in one of our isolates. Other ß-lactamase types (CTXM-15/4 isolates; SHV/2 isolates; OXA-48/3 isolates) were detected in association with New Delhi metallo-beta-lactamase (NDM) and/or Verona Integron-Mediated Metallo-ß-lactamase (VIM) enzymes. Furthermore, these isolates were resistant to other antimicrobial agents, including gentamicin [aac(3)-II/11 isolates], tetracycline (tetB or tetA/2 isolates), chloramphenicol (cmlA and/or floR/3 isolates), streptomycin (aadA/5 isolates), and sulfonamides (sul1 or sul2 or sul3/4isolates). The Multilocus Sequence Typing revealed 10 different Sequence types (ST) of which 7 novel ST: ST147 (3 isolates), ST101 (1 isolate), ST630 (1 isolate), ST3485 (1 isolate), ST3486 (1 isolate), ST3487 (1 isolate), ST3488 (1 isolate), ST3489 (1 isolate), ST3490 (1 isolate), ST3491 (2 isolates). Our study provides new data about MBL producing K. pneumoniae in Tunisia. Thus, we report for the first time the coexpression of blaNDM-1 and blaVIM-1 in our country and also we describe seven novel ST of MBL producing K. pneumoniae in the world.

Huge Diversity of TEM and SHV ß-Lactamases Types Among CTX-M-15-Producing Enterobacteriaceae Species in Tunisia.

The aim of our study was to characterize third-generation cephalosporin (3GC)-resistant Enterobacteriaceae isolated over two different periods from patients hospitalized in the Military Hospital of Tunis with special focus to class A ß-lactamases. This study included 180 Enterobacteriaceae resistant to 3GC isolated from samples of patients hospitalized in various services of the hospital. Enterobacteriaceae species detected by the Vitek 2 Compact® (BioMérieux®) automated system showed the dominance of Klebsiella pneumoniae followed by Escherichia coli during both periods. These strains were mainly isolated from urine samples and rectal swabs of patients hospitalized mostly in neonatology service and intensive care unit. The molecular research of genes encoding CTX-M, TEM, and SHV ß-lactamase types showed a high rate of strains producing CTX-M ß-lactamases, all of them harbored the blaCTX-M-15 gene. However, a huge diversity of SHV and TEM ß-lactamases types was discovered in our study. In fact, nine various subvariants of blaSHV gene (blaSHV-1, blaSHV-11, blaSHV-12, blaSHV-27, blaSHV-28, blaSHV-31, blaSHV-38, blaSHV-79, and blaSHV-81) and eight subvariants of blaTEM gene (blaTEM-70, blaTEM-71, blaTEM-76, blaTEM-77, blaTEM-79, blaTEM-105, blaTEM-148, and blaTEM-186) were identified among our Enterobacteriaceae species during both periods. All subvariants of blaTEM gene and some subvariants of blaSHV gene (blaSHV-31, blaSHV-38, blaSHV-79, and blaSHV-81) have not been previously detected in our country.

Vancomycin-Resistant Enterococcus faecium in Tunisia: Emergence of Novel Clones.

Vancomycin-resistant Enterococci (VRE) are a major public health problem worldwide, since they are commonly implicated in nosocomial infections in various regions in the world. The aim of our study was to investigate genetic features and clonal relationship of VRE in the Military hospital of Tunisia. A total of 10 VRE strains were initially detected and identified by the Viteck II compact® (BioMérieux®) automated system, then confirmed by PCR using specific primers. These VRE strains were isolated during the period extended between September 2015 and January 2017 from anal and blood samples from patients hospitalized mainly in the neonatology service and intensive care unit. All these strains were identified as Enterococcus faecium and carried the vanA gene. Other acquired resistance genes were also detected by PCR: [ermB (n = 6); tetL (n = 6); tetM (n = 2); aac(6')-Ie-aph(2'')-Ia (n = 10); aph(3')-III-a (n = 9); ant(6)-Ia (n = 8)]. The insertion sequence IS16 was detected in all our tested strains. Esp virulence gene was detected in only one strain. The clonal relatedness of VRE strains screened by pulse-field gel electrophoresis and multi-locus sequence typing showed four clones: two related clones A1 (one strain) and A2 (one strain) ascribed to ST80 belonged to CC17, the other remaining two clones, named B (one strain) and C (seven strains), revealed two new sequences types assigned to ST1463 and ST1464 respectively. The emergence of novel clones of VRE in this hospital could be a warning of rapid evolution of these resistant bacteria, which calls for new surveillance strategies, strict hygiene and practices.