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Background: Extended-spectrum β-lactamases (ESBLs), particularly CTX-M- type ESBLs, are among the most important resistance determinants spreading worldwide in Enterobacteriaceae. The aim of this study was to characterize a collection of 163 ESBL-producing Escherichia coli collected in Tunisia, their ESBL-encoding plasmids and plasmid associated addiction systems. Results: The collection comprised 163 ESBL producers collected from two university hospitals of Sfax between 1989 and 2009. 118 isolates harbored blaCTX-M gene (101 blaCTX-M-15 gene and 17 blaCTX-M-14 gene). 49 isolates carried blaSHV-12 gene, 9 blaSHV-2a gene and only 3 blaTEM-26 gene. 16 isolates produced both CTX-M and SHV-12. The 101 CTX-M-15-producing isolates were significantly associated to phylogroup B2 and exhibiting a high number of virulence factors. 24 (23.7%) of the group B2 isolates belonged to clonal complex ST131. Pulsed-field gel electrophoresis (PFGE) typing revealed a genetic diversity of the isolates. 144 ESBL determinants were transferable mostly by conjugation. The majority of plasmid carrying blaCTX-M-15 genes (72/88) were assigned to various single replicon or multireplicon IncF types and had significantly a higher frequency of addiction systems, notably the VagCD module. Conclusion: This study demonstrates that the dissemination of CTX-M-15 producing E. coli in our setting was due to the spread of various IncF-type plasmids harboring multiple addiction systems, into related clones with high frequency of virulence determinants. [ABSTRACT FROM AUTHOR]

Objectives To report an outbreak due to Providencia stuartii isolates carrying blaOXA-48, blaPER-1, blaCMY-4 and qnrA6 in a Tunisian hospital in 2011. Methods Eight intensive care unit (ICU) patients infected/colonized by extended-spectrum β-lactamase (ESBL)-producing P. stuartii between March and May 2011 were included. Molecular epidemiology was studied by PFGE. Antibiotic resistance genes were analysed by PCR and sequencing and the plasmid incompatibility group by a PCR-based replicon typing scheme. Results Eight patients were colonized with ESBL-producing P. stuartii isolates. All these isolates were clonally related and found to carry blaOXA-48, blaPER-1, blaCMY-4, qnrA6 and aac-6′-Ib genes on the same self-conjugative IncA/C plasmid. The same strain was also cultured from environmental samples in the ICU. All these isolates were susceptible to carbapenems. Only one colonized patient developed P. stuartii pleurisy and was effectively treated with imipenem alone. Conclusions This is the first report of an outbreak due to P. stuartii isolates carrying blaOXA-48 in Tunisia. The simultaneous expression of various resistance genes (blaOXA-48, blaCMY-4, blaPER-1, qnrA and aac-6′-Ib) by P. stuartii isolates is alarming. [ABSTRACT FROM PUBLISHER]

This study was conducted to identify the β-lactamase content of 30 metallo-β-lactamase-producing Pseudomonas aeruginosa isolated in 2007 from two Tunisian hospitals and to investigate their genetic relatedness. All these isolates produced VIM-2. blaPER-1, blaPSE-1, blaOXA-2, and blaOXA-10 were identified in 17, 5, 21, and 1 isolates, respectively. These enzymes were often associated in the same isolate: 26 isolates had at least two β-lactamases. The predominant serotype was O12. Pulsed-field gel electrphoresis revealed genetic diversity among the metallo-β-lactamase-producing P. aeruginosa isolates. This is the first report on the existence of blaPER-1, blaPSE-1, blaOXA-2, and blaOXA-10 in Tunisia. [ABSTRACT FROM AUTHOR]

Objectives: Escherichia coli producing CTX-M-type extended-spectrum β-lactamases (ESBLs) are spreading worldwide. The aim of this work was to investigate the addiction systems carried by the replicons involved in the emergence and spread of ESBLs in relation to ESBL and replicon types. [ABSTRACT FROM PUBLISHER]

A novel biocomposite with poly(methyl methacrylate) as resin containing Salvadora persica powders was elaborated. In this study, for the first time, S. persica was used to enhance the bioactive performance of dental restoration materials. Material characterization was carried out both on bare materials and on the elaborated biocomposite (30 wt% of S. persica). X-ray diffraction, Fourier transform infrared spectroscopy, differential scanning calorimetry, and high-performance liquid chromatography techniques were conducted to perform material characterization. The obtained results linked to dental material showed the presence of the organic chemical compounds of S. persica, which are responsible for biological activities, and the presence of mineral chemical compounds of S. persica, which are useful for dental applications and health. They also revealed the absence of toxic residual monomers. In addition, they proved the antioxidant activities proof of elaborated composite related to total polyphenol flavonoid content. Finally, they exhibited the antibacterial activity of S. persica and the composite. [ABSTRACT FROM AUTHOR]

Objectives: To determine the prevalence of community intestinal carriage of ESBL-producing Enterobacterales (ESBL-E), acquired-AmpC-producing Enterobacterales (aAmpC-E) and carbapenemase-producing Enterobacterales (CPE) in Tunisia.Methods: From November 2012 to September 2017, stool samples from food handlers in Sfax, Tunisia, were screened for ESBL-E, AmpC-E and CPE using antibiotic-containing media. The genes encoding these β-lactamases were characterized by PCR, sequencing, and transfer assays. ST131 clonal groups were detected by PCR and characterized for antibiotic resistance, virulence genes and PFGE patterns.Results: Of 2135 participants, ESBL-E, aAmpC-E, and CPE carriage were detected in 419 (19.63%), 35 (1.63%) and 7 (0.33%) participants, respectively. CTX-M-15 (60%), CTX-M-1 (16.8%) and CTX-M-27 (12.8%) were the most common ESBL determinants. The ESBL-E carriage was significantly higher in summer (33%) and autumn (25.7%) than in winter (12.1%) and spring (11.4%). ST131 was detected in 50 (13.2%) of the 378 ESBL-producing Escherichia coli isolates; most of them (35; 70%) belonged to subclade C1 (cluster C1-M27: 23 isolates, 46%; cluster C1-non-M27: 12 isolates, 24%) followed by those belonging to subclade C2 (11; 22%). Although subclade C2 isolates, all harbouring blaCTX-M-15, had the highest resistance rates and virulence factor and addiction system scores, the subclade C1 isolates, mainly harbouring blaCTX-M-27 (94%), were predominant since 2015. The most frequently detected carbapenemase-encoding gene was blaOXA-48-like (85%) and acquired AmpC-encoding genes were blaDHA-1 (54%) and blaCMY-2 (46%).Conclusions: This is the first large Tunisian study to reveal a high faecal ESBL carriage rate, a low CPE carriage rate, and the predominance of CTX-M-27-producing subclade C1 among faecal ESBL-ST131 isolates in the Tunisian community. [ABSTRACT FROM AUTHOR]

The article describes the spread of Klebsiella pneumoniae isolates producing OXA-48 β-lactamase at Sfax University Hospital in Tunisia. It reports the detection of 153 clinical isolates of K. pneumoniae with reduced susceptibility to extended-spectrum cephalosporins and/or imipenem. It suggests that the spread of blaOXA-48 gene is via diffusion of the same replicon in different clones or by localization of blaOXA-48 in a different replicon. It calls for a nationwide prevalence study of OXA-48-producing Enterobacteriaceae.

Summary: Objective: The aim of this study was to describe the epidemiological characteristics of Acinetobacter baumannii ventilator-associated pneumonia (VAP) and to identify factors predictive of a poor outcome. Methods: A retrospective study was conducted over 16 months in a Tunisian intensive care unit (ICU). All adult patients with A. baumannii VAP were included. Results: Ninety-two patients were included in they study; 41 (44.6%) were admitted because of multiple trauma. The mean age of the patients was 44.5±19.5 years. All patients needed mechanical ventilation on admission. The mean SAPS II score was 39±15. The mean delay before VAP onset was 8.1±4.7 days. On VAP onset, 57 patients (62%) developed septic shock. Only 14.2% of isolated strains were susceptible to imipenem; none were resistant to colistin. The mean duration of mechanical ventilation was 20±11 days. The mean duration of ICU stay was 24.3±18.7 days. ICU mortality was 60.9%. In the multivariate analysis, factors predictive of a poor outcome were previously known hypertension (odds ratio 5.8, 95% confidence interval 1.4–24.9; p =0.018) and VAP-related septic shock (odds ratio 8.5, 95% confidence interval 3–23.7; p <0.001). Conclusion: A. baumannii VAP is associated with a high mortality. Hemodynamic impairment is predictive of a poor outcome. [ABSTRACT FROM AUTHOR]

Background: Streptococcus pneumoniae remains a leading cause of morbidity and mortality worldwide. In this study, we sought to analyze serotype distributions, antibiotic resistance, and genetic relationships of 106 clinical invasive pneumococcal isolates recovered in Tunisia between 2012 and 2018, prior to the routine use of pneumococcal conjugate vaccines (PCV). Methods: We used multiplex PCR, the disk diffusion method and/or E-test, and multi-locus sequence typing (MLST). Results: The most frequent serotypes were 14 (17%), 19F (14.2%), and 3 (11.3%). Of the 106 S. pneumoniae isolates, 67.9% were penicillin non-susceptible (29.4% were resistant), 45.3% were amoxicillin non-susceptible (17% were resistant), and 16% were cefotaxime non-susceptible. For antibiotics other than β-lactams, resistance rates to erythromycin, tetracycline, cotrimoxazole, and chloramphenicol were 62.3, 33, 22.6, and 4.7%, respectively. Two isolates were non-susceptible to levofloxacin. Among 66 erythromycin-resistant pneumococci, 77.3% exhibited the cMLSB phenotype, and 87.9% carried ermB gene. All tetracycline-resistant strains harbored the tetM gene. The potential coverage by 7-, 10-, and 13-valent pneumococcal conjugate vaccines were 55.7, 57.5, and 81.1%, respectively. A multilocus sequence typing analysis revealed great diversity. Fifty different sequence types (STs) were identified. These STs were assigned to 10 clonal complexes and 32 singletons. The most common STs were 179, 2918, 386, and 3772 – related mainly to 19F, 14, 6B/C, and 19A serotypes, respectively. Conclusions: This study demonstrated that the majority of the serotypes of invasive pneumococci in the Tunisian population were 14, 19F, and 3. Moreover, we noted a high degree of genetic diversity among invasive S. pneumoniae isolates. The highest proportions of antibiotic non-susceptible isolates were for penicillin, erythromycin, and tetracycline. Further molecular characteristics are required to monitor the genetic variations and to follow the emergence of resistant pneumococci for the post-vaccination era in Tunisia. [ABSTRACT FROM AUTHOR]

Streptococcus pneumoniae remains a significant cause of morbidity and mortality worldwide despite the overall success of the vaccine programs. In Tunisia, pneumococcal conjugate vaccines (PCV)10 was introduced in the national immunization program in April 2019. We sought to determine the relationship between serotypes and antimicrobial nonsusceptibility of S. pneumoniae isolates recovered from clinical samples in the prevaccination period in the south of Tunisia. A total of 504 nonduplicate S. pneumoniae isolates collected between 2012 and 2018 were tested for antimicrobial susceptibility, among them 439 (87.1%) were serotyped. The most common serotypes were 19F (17.8%), 14 (15.3%), 3 (9.1%), 19A (8.2%), and 23F (7.3%). The proportions of isolates with serotypes covered by PCV7, PCV10, and PCV13 were 55.4%, 56.3%, and 77.9%, respectively. Three-quarters (74.4%) of pneumococcal isolates were nonsusceptible to penicillin, and about half (54.8%) were multidrug resistant. Penicillin nonsusceptibility was observed for all 19A and 23F isolates, and was significantly associated with serotypes 19F (odds ratio [OR]: 33.7) and 14 (OR: 8.7). A significant association with multidrug resistance was noted for serotypes 19A (OR: 10), 19F (OR: 9.4), 23F (OR: 8.6), and 6B (OR: 5.2). The alarming rates of pneumococcal antimicrobial nonsusceptibility and the strong association with the most prevalent serotypes compel microbiologists to monitor the impact of the PCV10 introduced recently in our national immunization program. [ABSTRACT FROM AUTHOR]

Introduction: Streptococcus pneumoniae can be responsible for severe human infections. Optochin resistance has been a potential cause of misidentification of pneumococcus and other members of the mitis group. Hence, rapid and easy optochin resistant (Optr) S. pneumoniae identification is essential. Methodology: Atypical pneumococci were characterized using optochin susceptibility, bile solubility based on spectrophotometric reading, serotyping, pulsed field gel electrophoresis (PFGE), 16S rRNA sequencing and PCR-based assays targeting pneumococcal genes lytA, ply, pspA, cpsA, Spn9802 and Spn9828. Results: Optical density values for the bile solubility test suggest the identification of four Optr S. pneumoniae and one Streptococcus pseudopneumoniae. All Optr pneumococci harbored cpsA, lytA, ply, Spn9802, Spn9828 and pspA genes. Only ply, spn9802 and Spn9828 genes were detected in S. pseudopneumoniae. The 16S rRNA sequencing differentiates between these two species. Optr S. pneumoniae strains belonged to different genotypes and serotypes (14, 19A, 3 and 9V). Three Optr S. pneumoniae isolates were typed as pspA family 2, while one belonged to pspA family 1. Sequencing of the atpA and atpC gene of the Optr variants revealed three mutations in the ATPase a-subunit (L99I, M23V and V52I) and one mutation in ATPase c-subunit (V48I). Conclusions: Our data indicate that bile OD-values provides an accurate, fast and easy method to discriminate between Optr S. pneumoniae and other Streptococcus mitis group. Moreover molecular techniques, confirming the bile test, can be used in order to prevent these atypical pneumococci and alert clinical microbiologists of the presence of these strains in the community. [ABSTRACT FROM AUTHOR]

The treatment of acute osteomyelitis is becoming more challenging since the emergence of community-acquired methicillin-resistant Staphylococcus aureus. We collected data on all patients with acute osteomyelitis caused by this germ over a period of 21 years (January 1995- December 2016) and we analyzed the peculiarities of this disorder. Our case series includes 15 children, with an average age of 9 years. All patients had affected lower limb. Local trauma was reported in 8 cases and skin carriage in 4 cases. Acute onset was reported in 12 cases associated with pseudoparalysis of the affected limb. One patient had Staphylococcus aureus pulmonary infection with signs of septicopyemia. Blood culture was positive in 8 cases. In one case PCR assay for detection of Panton-Valentine leukocidin was performed with positive result. All these patients underwent surgical debridement and received secondarily adapted empirical antibiotic therapy. Outcome was good in 8 cases and poor in the other cases, with transition to a chronic state in 6 cases and one case of death. Pathological fracture was reported in 3 cases. Osteomyelitis cause by community-acquired methicillin-resistant Staphylococcus aureus is associated with a pejorative outcome. Recognizing the clinical and paraclinical signs of these infections is essential for a specific and early therapeutic management. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

The article discusses research which investigated outcomes associated with the combined use of colistin and tigecycline or colistin and imipenem/cilastatin for the treatment of Acinetobacter baumannii (Ab)-related ventilator-acquired pneumonia (VAP). Topics explored include the intensive care unit (ICU) admission of patients involved in the study, the drug dosage and combination regimens administered to patients, and the Ab bloodstream infections noted following treatment.

Objectives To analyze the serotype distribution of Streptococcus pneumoniae clinical isolates collected in the south of Tunisia over a 5-year period in different age groups and to assess their antimicrobial susceptibility patterns. Methods A total of 305 non-duplicate S . pneumoniae isolates were collected between January 2012 and December 2016 at the university hospital in Sfax, Tunisia. All isolates were serotyped by multiplex PCR. The antibiotic susceptibility of all isolates was determined using the disk diffusion test or Etest assay. Results Among the 305 pneumococcal isolates, 76 (24.9%) were invasive and 229 (75.1%) were non-invasive. The most common serotypes were 19F (20%), 14 (16.7%), 3 (9.2%), 23F (7.5%), 19A (5.9%), and 6B (5.9%). Potential immunization coverage rates for pneumococcal conjugate vaccines PCV7, PCV10, and PCV13 were 58%, 59.3%, and 78.7%, respectively. Three-quarters (75.3%) of pneumococcal isolates were non-susceptible to penicillin. The resistance rate to erythromycin was 71.4%. Only two isolates were resistant to levofloxacin. Conclusions 19F and 14 were the most prevalent serotypes in the south of Tunisia. The inclusion of a PCV in the immunization program could be useful for reducing the burden of pneumococcal diseases. The high resistance rate to penicillin and macrolides is alarming. Prudent use of antibiotics is crucial to prevent the selection of multidrug-resistant pneumococci. [ABSTRACT FROM AUTHOR]

Enteritidis, Typhimurium and Livingstone are the main Salmonella enterica serovars recovered in Tunisia. Here, we aimed to assess the genetic diversity of fifty-seven Salmonella enterica strains from different sampling periods, origins and settings using pulsed-field gel electrophoresis (PFGE), multi-locus sequence typing (MLST) and multi-locus variable-number tandem repeat analysis (MLVA). Salmonella Enteritidis, isolated from human and food sources from two regions in Sfax in 2007, were grouped into one cluster using PFGE. However, using MLVA these strains were divided into two clusters. Salmonella Typhimurium strains, recovered in 2012 and represent sporadic cases of human clinical isolates, were included in one PFGE cluster. Nevertheless, the MLVA technique, divided Salmonella Typhimurium isolates into six clusters with diversity index reaching (DI = 0.757). For Salmonella Livingstone which was responsible of two nosocomial outbreaks during 2000-2003, the PFGE and MLVA methods showed that these strains were genetically closely related. Salmonella Enteritidis and Salmonella Livingstone populations showed a single ST lineage ST11 and ST543 respectively. For Salmonella Typhimurium, two MLST sequence types ST19 and ST328 were defined. Salmonella Enteritidis and Salmonella Typhimurium strains were clearly differentiated by MLVA which was not the case using PFGE. [ABSTRACT FROM AUTHOR]

The article discusses research on ciprofloxacin-resistant Salmonella enterica serotype Kentucky ST198 isolates producing carbapenemases. Topics addressed include two carbapenemases-producing ciprofloxacin-resistant Kentucky ST198 clinical isolates acquired from two northern African countries, and the prophylactic use of fluoroquinolones in African and Asian poultry flocks as the main cause of the rapid spread of the ciprofloxacin-resistant Kentucky ST198 strain.

Typhimurium is one of the main Salmonella serovar responsible for non-typhoidal gastro-enteritis in Tunisia. Here, we aimed to assess the genetic diversity of 88 clinical Salmonella Typhimurium strains recovered during 14 years from 2000 to 2013. Phage typing, CRISPR polymorphisms (CRISPOL), pulsed-field gel electrophoresis (PFGE), multi-locus variable-number tandem repeat analysis (MLVA) and Whole genome sequencing (WGS) were used to study the relatedness and spatio-temporal evolution of Salmonella Typhimurium populations (Typhimurium (n = 81), monophasic (n = 3) and nonmotile (n = 4) variants). Seven-locus MLST from whole genome assemblies showed that all isolates, except one, belonged to ST19. The isolates were divided into 10 definitive phage (DT) types, dominated by DT104-L (39.8%), DT41 (14.8%), DT116 (11.4%) and DT120 (5.7%). Fifty-seven MLVA patterns (DI, 0.978) were obtained compared to 11 different CRISPOL types and 15 PFGE types (DI,0.845). For cgMLST analysis, 20 profiles were found. A total of 3056 SNPs were identified from the whole genome of the 88 Salmonella Typhimurium isolates. These SNPs resolved these isolates into 86 SNP haplotypes. The phylogeny result allocated most Salmonella Typhimurium isolates into four distinct clades and seven subclades. Genetic diversity between the four clades ranged in the order of 249 to 720 nucleotide changes. The prevalent phage type DT104L formed a major clade on the phylogenetic tree. Pairwise SNP differences between the strains of this clade ranged between 0 and 59. SNP-based WGS typing seems to be the most valuable molecular markers for studying the evolutionary relationships of homogeneous serovar Typhimurium isolates. • This is the first study of Salmonella Typhimurium strains isolated in Tunisia that used WGS to access the genetic diversity. • WGS technique gives additional resolution to differentiate between the homogenous strains using SNPs analysis. • This is the first identification of phage type of Salmonella Typhmurium circulating in Tunisia from 2000 to 2013. [ABSTRACT FROM AUTHOR]