Nasopharyngeal Carriage of Streptococcus pneumoniae in Tunisian Healthy under-Five Children during a Three-Year Survey Period (2020 to 2022).

We aimed to assess the prevalence of nasopharyngeal pneumococcal carriage and to determine serotype distribution, antibiotic susceptibility patterns, and evolutionary dynamics of Streptococcus pneumoniae isolates in healthy under-five children. Nasopharyngeal swabs were collected from healthy children over three survey periods between 2020 and 2022. All pneumococcal isolates were serotyped and tested for antimicrobial susceptibility. A total of 309 S. pneumoniae isolates were collected, with an overall prevalence of nasopharyngeal pneumococcal carriage of 24.4% (CI95%: [22-26.8%]). These isolates were classified into 25 different serotypes. The most common serotypes were 14 (14.9%), 19F (12%), 6B (10.4%), and 23F (7.4%), which are covered by the PCV10 vaccine, as well as 19A (8.4%) and 6A (7.8%), which are covered by the PCV13 vaccine. A significant decrease in the proportion of serotype 19F (p = 0.001) and an increase in serotypes 19A (p = 0.034) and 6A (p = 0.029) were observed between the three survey periods. Multidrug resistance (MDR) was noted for 56.6% of the isolates. A significant association with antimicrobial resistance was observed for the most frequent serotypes, mainly serotype 19A. In conclusion, one-quarter of healthy under-five children in Tunisia carried S. pneumoniae in their nasopharynx. A dominance of vaccine serotypes significantly associated with antimicrobial resistance was recorded.

Group B Streptococcus and Perinatality in the South of Tunisia: Epidemiology, Serotype Distribution, and Antibiotic Susceptibility.

INTRODUCTION: In the lack of updated Tunisian epidemiological data, we sought to describe the epidemiology of Group B Streptococcus (GBS) in pregnant women and newborns. MATERIALS AND METHODS: A retrospective analysis of GBS neonatal invasive infections and a cross-sectional study evaluating the prevalence of maternal GBS colonization were conducted. GBS isolates were tested for antimicrobial susceptibility, serotyped, and assessed for the appurtenance to the hypervirulent ST17 clone. RESULTS: Of 98 neonates with GBS, early-onset GBS disease (EOD) comprised 83.7 and 16.3% were late-onset GBS disease (LOD). The prevalence of maternal GBS colonization was 27%. All GBS isolates were susceptible to penicillin. Serotype III predominated (42.6%) for neonatal invasive infections. GBS isolates belonging to the ST17 sequence type were found only as serotype III. CONCLUSION: This study documents the frequency of GBS EOD, the high rate of maternal GBS colonization, and the predominance of the hypervirulent clone type III/ST17 in infants.

Antibiotic resistance pattern, capsular types, and molecular characterization of invasive isolates of Streptococcus pneumoniae in the south of Tunisia from 2012 to 2018.

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 ß-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.

Antimicrobial resistance genes, virulence markers and prophage sequences in Salmonella enterica serovar Enteritidis isolated in Tunisia using whole genome sequencing.

Salmonella Enteritidis causes a major public health problem in the world. Whole genome sequencing can give us a lot of information not only about the phylogenetic relatedness of these bacteria but also in antimicrobial resistance and virulence gene predictions. In this study, we analyzed the whole genome data of 45 S. Enteritidis isolates recovered in Tunisia from different origins, human, animal, and foodborne samples. Two major lineages (A and B) were detected based on 802 SNPs differences. Among these SNPs, 493 missense SNPs were identified. A total of 349 orthologue genes mutated by one or two missense SNPs were classified in 22 functional groups with the prevalence of carbohydrate transport and metabolism group. A good correlation between genotypic antibiotic resistance profiles and phenotypic analysis were observed. Only resistant isolates carried the respective molecular resistant determinants. The investigation of virulence markers showed the distribution of 11 Salmonella pathogenicity islands (SPI) out of 23 previously described. The SPI-1 and SPI-2 genes encoding type III secretion systems were highly conserved in all isolates except one. In addition, the virulence plasmid genes were present in all isolates except two. We showed the presence of two fimbrial operons sef and ste previously considered to be specific for typhoidal Salmonella. Our collection of S. Enteritidis reveal a diversity among prophage profiles. SNPs analysis showed that missense mutations identified in fimbriae and in SPI-1 and SPI-2 genes were mostly detected in lineage B. In conclusion, WGS is a powerful application to study functional genomic determinants of S. Enteritidis such as antimicrobial resistance genes, virulence markers and prophage sequences. Further studies are needed to predict the impact of the missenses SNPs that can affect the protein functions associated with pathogenicity.

Relationship Between Serotypes and Antimicrobial Nonsusceptibility of Streptococcus pneumoniae Clinical Isolates in Tunisia.

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.

Clinical optochin resistant Streptococcus pneumoniae and Streptococcus pseudopneumoniae strains in Tunisia.

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.

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 diversity of clinical Salmonella enterica serovar Typhimurium in a university hospital of south Tunisia, 2000-2013.

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.

Serotype distribution and antibiotic susceptibility of Streptococcus pneumoniae strains in the south of Tunisia: A five-year study (2012-2016) of pediatric and adult populations.

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.

Isolation and characterization of a new Bacillus thuringiensis strain with a promising toxicity against Lepidopteran pests.

Insecticides derived from Bacillus thuringiensis are gaining worldwide importance as environmentally desirable alternatives to chemicals for the control of pests in public health and agriculture. Isolation and characterization of new strains with higher and broader spectrum of activity is an ever growing field. In the present work, a novel Tunisian B. thuringiensis isolate named BLB459 was characterized and electrophoresis assay showed that among a collection of 200 B. thuringiensis strains, the plasmid profile of BLB459 was distinctive. SmaI-PFGE typing confirmed the uniqueness of the DNA pattern of this strain, compared with BUPM95 and HD1 reference strains. PCR and sequencing assays revealed that BLB459 harbored three cry genes (cry30, cry40 and cry54) corresponding to the obtained molecular sizes in the protein pattern. Interestingly, PCR-RFLP assay demonstrated the originality of the BLB459 cry30-type gene compared to the other published cry30 genes. Insecticidal bioassays showed that BLB459 spore-crystal suspension was highly toxic to both Ephestia kuehniella and Spodoptera littoralis with LC50 values of about 64 (53-75) and 80 (69-91) µg of toxin cm(-2), respectively, comparing with that of the commercial strain HD1 used as reference. Important histopathological effects of BLB459 δ-endotoxins on the two tested larvae midguts were detected, traduced by the vacuolization of the apical cells, the damage of microvilli, and the disruption of epithelial cells. These results proved that BLB459 strain could be of a great interest for lepidopteran biocontrol.

Molecular epidemiological characteristics of Salmonella enterica serovars Enteritidis, Typhimurium and Livingstone strains isolated in a Tunisian university hospital.

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.

Nosocomial dissemination of Providencia stuartii isolates carrying bla OXA-48, bla PER-1, bla CMY-4 and qnrA6 in a Tunisian hospital.

OBJECTIVES: To report an outbreak due to Providencia stuartii isolates carrying bla(OXA-48), bla(PER-1), bla(CMY-4) and qnrA6 in a Tunisian hospital in 2011. METHODS: Eight intensive care unit (ICU) patients infected/colonized by extended-spectrum ß-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 bla(OXA-48), bla(PER-1), bla(CMY-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 bla(OXA-48) in Tunisia. The simultaneous expression of various resistance genes (bla(OXA-48), bla(CMY-4), bla(PER-1), qnrA and aac-6'-Ib) by P. stuartii isolates is alarming.

Diversity of ß-lactamases in Pseudomonas aeruginosa isolates producing metallo-ß-lactamase in two Tunisian hospitals.

This study was conducted to identify the ß-lactamase content of 30 metallo-ß-lactamase-producing Pseudomonas aeruginosa isolated in 2007 from two Tunisian hospitals and to investigate their genetic relatedness. All these isolates produced VIM-2. bla(PER-1), bla(PSE-1), bla(OXA-2), and bla(OXA-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 ß-lactamases. The predominant serotype was O12. Pulsed-field gel electrophoresis revealed genetic diversity among the metallo-ß-lactamase-producing P. aeruginosa isolates. This is the first report on the existence of bla(PER-1), bla(PSE-1), bla(OXA-2), and bla(OXA-10) in Tunisia.

Molecular epidemiology and genetic environment of acquired bla ACC-1 in Salmonella enterica serotype Livingstone causing a large nosocomial outbreak in Tunisia.

Eighty-four isolates of Salmonella enterica serovar Livingstone were collected from patients hospitalized in a pediatric ward in Sfax Hospital (South Tunisia). These isolates were responsible for two nosocomial outbreaks in 2000 and 2002. Twenty-eight clinical isolates of S. enterica serovar Livingstone were also obtained in two other Tunisian hospitals in Monastir (Central Tunisia) and Tunis (North Tunisia), respectively, in 2002 and 2003. Pulsed-field gel electrophoresis yielded that these isolates were closely related. Antimicrobial susceptibility testing showed a particular beta-lactam resistance phenotype, suggestive of the presence of an AmpC-type enzyme in 111 of the 112 clinical isolates. bla(ACC-1) was characterized by polymerase chain reaction (PCR) and sequence analysis in the 111 isolates. TEM-1 was characterized in all strains and SHV-2a in only two strains. The genetic organization of bla(ACC-1) was determined by PCR mapping and sequencing. The plasmid-borne bla(ACC-1) gene mapped immediately downstream from ISEcp1. This ISEcp1 insertion sequence was itself disrupted by IS26 insertion sequences. A supplementary deletion of 13 bp was observed in ISEcp1 upstream IS26, in all isolates from Tunis, except one. PCR analysis and sequencing also revealed the presence of tnpR, bla(SCO-1), gdha, IS1353, and TniB Delta 1.

[Spinal cord compression due to benign osteoporotic vertebral fracture]. / Compression médullaire par un tassement vertébral ostéoporotique bénin.

BACKGROUND: A neurologic compromise associated with vertebral fractures is generally due to a malignancy causes. Therefore, an osteoporotic vertebral fracture can sometimes cause neurologic complications. AIM: Report a case of neurologic compromise associated with osteoporotic vertebral fractures. OBSERVATION: A-62-year-old man suffered from rheumatoid arthritis since 1985, presented a cervical pain associated with quadriparesia secondary to a C5 osteoporotic vertebral fractures. CONCLUSION: Osteonecrosis may be the cause of neurologic compromise associated with osteoporotic vertebral fractures.

[Naso-sinusienne sarcoidosis]. / La sarcoïdose naso-sinusienne.

BACKGROUND: Sarcoidosis is an ubiquity disease, which can infiltrate all tissues. The cutaneous and ophthalmologic thoracic localizations are most frequent. The nasosinusienne localisation is rare. AIM: The authors report their observations of 4 patients: 2 men and 2 women with a mean age 47.5 years (42-56). Theses cases was diagnosed and treated between january 1998 and December 2003 in the ENT service of the Habib Thameur hospital. CASES: The diagnosis was related to a nasal or a sinuses biopsy. The assessment of extension was negative in 3 cases. The corticoid treatment in local pulverization was sufficient in 2 cases. The corticoid treatment by systematic way was necessary among 2 patients. Favourable out look was obtained in all the cases. The naso sinusienne localization is rare, it is exceptionally isolated. The clinical and radiological symptoms are not specifics. Principal element of the diagnosis is the directed biopsy, easy in this localization.