Staphylococcus aureus Ventilator-Associated Pneumonia: A Study of Bacterio-Epidemiological Profile and Virulence Factors.

Ventilator-associated pneumonia (VAP) represents a major cause of nosocomial infections in the intensive care units in which Staphylococcus aureus is frequently involved. Better knowledge of this pathogen is required in order to enhance the patient's treatment and care. In this article, we studied the bacteriological profile and virulence factors of S. aureus-related VAP on a 3-year period. We included a collection of S. aureus strains (n = 35) isolated from respiratory samples from patients diagnosed with VAP in the intensive care units. We studied the bacteriological aspects and we searched for the presence of virulence factors (SpA, FnbpA, Hla, and PVL genes) in the strains, and we also studied the clinical and biological aspects of the infections. The average age of our patients was of 36 years and they were predominantly males (sex ratio = 3.37). A severe head trauma or a history of coma was noted in 73.43% of the patients. The average duration of ventilation was 29 days. Among the studied strains, five were Methicillin-resistant S. aureus of which three expressed the mecA gene. Overall, the Hla gene was detected in 85.7% of the strains and it was more prevalent in Methicillin-susceptible than Methicillin-resistant strains (93.3% versus 40%; P = 0.014). FnbpA, Spa, and PVL genes were detected, respectively, in 80%, 45.7%, and 20% of the strains. Therefore, our studied strains were essentially associated with the production of Hla and FnbpA genes. It is, however, important to elucidate their expression in order to establish their role in the VAP pathogenesis.

Elizabethkingia meningoseptica: An Uncommon Cause of Peritonitis In Continuous Ambulatory Peritoneal Dialysis: A Case Report and Review of Literature.

Peritonitis is a major complication of peritoneal dialysis (PD) and due to its gravity, it remains the primary reason to switch from PD to hemodialysis. Elizabethkingia meningoseptica, a non-fermentative Gram-negative bacillus, is rarely encountered as a pathogen causing peritonitis in adults. We present here a case report of an acquired infection with this organism in adult on PD. To the best of our knowledge, this is the first report of infection with this organism in a continuous ambulatory PD patient in Tunisia.

Unexpected predominance of rotavirus G9P[8] strain in Tunisian adult diarrheal patients.

Introduction. Group A Rotavirus (RVA) is known to be a major cause of acute gastroenteritis (AGE) in children but its role as a potential pathogen in immunocompetent adults is probably underestimated.Aim. To compare RVA infections in patients from different age groups.Methodology. Fecal samples were collected from patients aged from birth to 65 years, hospitalized or consulting for AGE between 2015 and 2017. All samples were screened by RT-PCR for the detection of VP6 gene specific of RVA. RVA-positive samples were VP7 and VP4 genotyped using multiplex semi-nested RT-PCR. Full-length VP7 gene of G9-positive strains were sequenced and submitted for phylogenetic analysis.Results. Of 1371 stool specimens collected from children (<5 years; n=454), older children (5 to <15 years; n=316) and adults (15-65 years; n=601), 165 (12.0 %) were RVA-positive. RVA detection rates were significantly higher in children and adults than in older children (15.8 % and 12.1 Vs 6.3 %, respectively; P<0.001). While RVA infections were mostly detected during the coldest months in children, they were observed all year-round in patients aged >5 years. Although G1P[8] remained the most prevalent combination (41.7 %) detected in children, G9P[8] strains widely predominated in adults (58.1 %), followed by G2P[4] (12.9 %). All characterized G9 strains clustered in the modern lineage III.Conclusion. RVA play an important role in AGE not only in children but also in adults. The findings of a wide G9 predominance in patients >5 years highlights the need for continuing surveillance in both pediatric and mature populations.

Dynamics and molecular features of OXA-48-like-producing Klebsiella pneumoniae lineages in a Tunisian hospital.

OBJECTIVES: The aim of this study was to elucidate the molecular features of genes, plasmids and clones of OXA-48-like producingKlebsiella pneumoniae isolates recovered in Sahloul Hospital (Sousse, Tunisia) in the period 2012-2014. METHODS: In vitro antimicrobial susceptibility testing, S1 nuclease pulsed-field gel electrophoresis (S1-PFGE), Southern blotting and PCR-based replicon typing (PBRT) were performed. Extended-spectrum ß-lactamase (ESBL) and carbapenemases genes were detected by PCR and sequencing. The clonality of isolates was assessed by PFGE and multilocus sequence typing (MLST). RESULTS: Klebsiella pneumoniae accounted for 26.8% (1095/4083) of clinical Enterobacterales isolates identified during 2012-2014, of which 21.9% (240/1095) were resistant to carbapenems, mostly harbouring blaOXA-48-like genes (196/240; 81.7%). Plasmid analysis showed that blaOXA-204 and blaOXA-48 were mostly carried by IncA/C and IncL plasmids, respectively. The current data highlight the dominance of two ST101 and ST147 lineages spreading OXA-48 and OXA-204, respectively, through successive clonal spreads at this hospital. In addition, a large diversity of other K. pneumoniae lineages was also identified, such as ST15, ST36 and ST525 spreading OXA-48 as well as ST340, ST2032, ST301, ST199 and ST1561 spreading OXA-48 or OXA-204, constituting a reservoir of possible dominant clones in the future. CONCLUSION: This study reports the full molecular characterisation of carbapenem resistance in K. pneumoniae and the predominance of a few clones responsible for the dissemination of OXA-48 and OXA-204 enzymes in a Tunisian hospital.

Evaluation of antibody persistence after a four-dose primary hepatitis B vaccination and anamnestic immune response in children under 6 years.

Introduction. Tunisia is an intermediate hepatitis B virus (HBV) endemic country. The vaccination against hepatitis B was introduced in 1995 including four doses with a first dose administrated at birth. Decreasing the level of antibodies against hepatitis B surface antigen (anti-HBs) over time can be alarming. This study was conducted to explore the anti-HBV immune response among children under 6 years old, vaccinated according to the national vaccination schedule, by evaluating the immunological response to primary vaccination and by exploring the anamnestic immune response to a booster dose.Methods. We conducted a cross-sectional prospective study from June 2016 to June 2017 (n=180), based on voluntary participation. Children were recruited from the public pediatric ward sectors in Sahloul University Hospital of Sousse in Central Tunisia. An anti-HB titre was determined based on electro-chemiluminescence micro-particle immunoassay (ECLIA), using Elecsys Anti-HBs II kit, Roche.Results. Mean age at the time of enrollment in the study was 33±14.8 months. The seroprotection rate was 77.2 %. The anti-HB titre differed significantly between the different age groups (P=0.002). The predicting variable for having no seroprotective antibody level was older age. Children with anti-HB levels <10 IU l- 1 were offered an additional dose of HBV vaccine. Anamnestic response 1 month after the challenge dose was observed in 100 % of subjects. The probability of developing a high antibody response, following the booster dose increased in conjunction with an increased pre-booster antibody level.Conclusion. The response to a booster dose suggests the persistence of immune memory in almost all vaccinated individuals. Although a booster dose increases substantially anti-HB titre, the clinical relevance of such an increase remains unknown.

Epidemiology of resistance and phenotypic characterization of carbapenem resistance mechanisms in Klebsiella pneumoniae isolates at Sahloul University Hospital-Sousse, Tunisia.

OBJECTIVE: To assess the prevalence of ESBL producing and carbapenem resistant Klebsiella pneumoniae isolated from in-come and out-come patients at Sahloul-university hospital. METHODS: A retrospective study over a 3 years period (January 2012 and December 2014) focused on 2160 strains of Klebsiella pneumoniae. Statistical analysis was carried out using SPSS program. ESBL detection was performed using a double disc diffusion method and carbapenemase detection was realized by Rosco-Disk kit. RESULTS: A total of 2160 Klebsiella pneumoniae strains were isolated during the period of the study, 26.2% (n=566) were ESBL-producers and 15.8% (n=342) showed resistance to carbapenem. The wards most affected by these strains were basically urology and intensive care units. Eighty four percent of studied strains (203/241) were resistant to temocillin, which correlate with the production of a class D (OXA-48-like) carbapenemase and 7% (17/241) showed sensitivity to EDTA and dipicolinic acid, which indicate the production of metallo-enzyme. The rate of resistance to colistin remains low. CONCLUSION: Resistance of Enterobacteriaceae, including K. pneumoniae, to third generation cephalosporins (3rd GC) and carbapenem through the mechanism of ESBL and carbapenemases production is becoming increasingly worrying. This suggests a more rational use of antibiotics, as well as the rigorous application of hygiene measurement.

Molecular characterization of group A rotavirus among children aged under 5 years in Tunisia, 2015-2017.

The aim of the present study was to report the molecular characterization of human group A rotaviruses (RVAs) circulating in Tunisia. Stool specimens were collected from children under 5 years of age who had been hospitalized or were consulting for gastroenteritis in Tunisian hospitals between 2015 and 2017. All samples were screened by reverse-transcription polymerase chain reaction (RT-PCR) for the detection of the VP6 gene specific for RVA. RVA-positive samples were further analysed for G/P genotyping by semi-nested multiplex RT-PCR. Among 454 tested samples, 72 (15.8 %) were positive for RVA. G1P[8] was the most prevalent detected strain (41.7%), followed by G9P[8] (32.8%), G2P[4] (7.5%), G12P[8] (7.5%), G1P[6] (3.0%), G2P[8] (1.5%) and G3P[8] (1.5%), with mixed infections in 4.5 % of cases. In the absence of a national anti-rotavirus vaccination strategy, RVAs remain the primary aetiological agent for gastroenteritis in Tunisian children.

Epidemiology and Whole-Genome Analysis of NDM-1-Producing Klebsiella pneumoniae KP3771 from Tunisia.

Objectives: The whole-genome sequence (WGS) of Klebsiella pneumoniae KP3771 isolate was characterized. This strain was recovered from the urine sample of an 80-year-old man hospitalized in an intensive care unit of the University Hospital Tahar Sfar in Tunisia. Materials and Methods: WGS using a MiSeq platform was used. The assembled genome was subjected to several software analyses. Results: K. pneumoniae KP3771 was resistant to all antibiotics but colistin and tigecycline. WGS analysis found 18 transmissible genes encoding resistance markers, including blaNDM-1 and blaCTX-M-15 genes, which were carried by four plasmids belonging to the Inc Ib, IIk, and R groups. Three families of genes encoding virulence factors were detected, including adhesins (fimH, fimA, fimB, fimC, mrkD, Kpn, and ycfM), siderophores (enterobactin, aerobactin, and yersiniabactin siderophores), and protectin/invasin (traT). The strain was assigned to the sequence type 147. Conclusions: This study describes the genome of a carbapenemase-producing K. pneumoniae clinical isolate recovered in Tunisia. Bacteria WGS has become the reference technology to address epidemiological issues; this high level of information is particularly well suited to enrich epidemiological workflows' output.

Whole-genome sequencing of NDM-1-producing ST85 Acinetobacter baumannii isolates from Tunisia.

BACKGROUND: New Delhi metallo-ß-lactamase (NDM)-producing Acinetobacter baumannii have been described in several countries worldwide, and studies have suggested that Acinetobacter spp. could play the role of intermediate progenitor of the blaNDM-1 gene between environmental progenitor and Enterobacteriaceae. MATERIALS AND METHODS: In total, 246 carbapenem-resistant A. baumannii isolates from a teaching hospital in Sousse, Tunisia were investigated between 1st June 2013 and 31st December 2015 to detect metallo-ß-lactamase (MBL) production. Polymerase chain reaction (PCR), antibiotic susceptibility testing, and genetic and whole-genome sequencing tools were used to study the underlying carbapenem resistance mechanisms. RESULTS: PCR screening of the 246 carbapenem-resistant A. baumannii isolates revealed that 242 of 246 isolates harboured carbapenemase genes (seven of 246 positive for blaNDM-1, four of 246 positive for blaNDM-1 and blaOXA-23, 231 positive for blaOXA-23). Conjugation and electroporation experiments suggested that the blaNDM-1 gene is likely to be chromosomally located. All the NDM-1-producing A. baumannii isolates were clonally related, and belonged to ST85 according to the Pasteur Institute's multi-locus sequence typing scheme. Analysis of the immediate genetic environment of the blaNDM-1 gene revealed that the gene was located within a truncated isoform of Tn125 transposon (ΔTn125). The blaOXA-23 gene was located within transposon Tn2008. CONCLUSION: This study showed the dissemination of a single clone of NDM-1-producing A. baumannii in a Tunisian hospital. Countries in north Africa may constitute a significant reservoir for NDM-1-producing A. baumannii. The spread of the blaNDM-1 gene in A. baumannii was linked to clonal spread in this study.

Genomic analysis of in vivo acquired resistance to colistin and rifampicin in Acinetobacter baumannii.

Acinetobacter baumannii is an opportunistic pathogen in healthcare facilities responsible for nosocomial infections mostly in immunocompromised patients. Colistin resistance is increasingly reported worldwide in A. baumannii. Here we describe the in vivo selection of colistin and rifampicin resistance in carbapenem-resistant A. baumannii. Antimicrobial susceptibility testing, plasmid analysis and whole-genome sequencing (WGS) were performed to fully characterise the resistome of two clinical isolates (AbS1 and AbS2) selected during treatment. Clinical isolate AbS1 remained susceptible to colistin, rifampicin and tigecycline, whilst AbS2 was susceptible only to tigecycline. PCR analysis revealed the presence of a blaOXA-23-like carbapenemase gene. Kieser extraction revealed an ca. 74 kb plasmid harbouring blaOXA-23. WGS revealed genomes of 3.8 Mbp in size with a G + C content of 38.9%, and both belonged to ST281 according to the Oxford MLST scheme and ST641 according to the Institut Pasteur scheme. The resistome was also composed of naturally occurring ß-lactamases, i.e. ADC-25 cephalosporinase and OXA-82 oxacillinase, aminoglycoside resistance genes [aac(3)-Ia, aadA1 and aph(3')-VIa (aphA6)], and mutations in DNA gyrases explaining fluoroquinolone resistance. Single nucleotide polymorphism analysis revealed that both isolates were identical except for a 30-nucleotide duplication within the pmrB gene and a point mutation in the rpoB gene resulting in colistin and rifampicin resistance, respectively. This study highlights the genomic plasticity of A. baumannii under antibiotic pressure. The 10-amino acid duplication in PmrB affects colistin susceptibility by regulating lipopolysaccharide modification through the PmrAB two-component system. These findings provide further information on the molecular mechanisms leading to colistin resistance in A. baumannii.

Genomic Insights into Colistin-Resistant Klebsiella pneumoniae from a Tunisian Teaching Hospital.

The emergence of colistin-resistant Klebsiella pneumoniae (CoRKp) is a public health concern, since this antibiotic has become the last line of treatment for infections caused by multidrug-resistant (MDR) Gram negatives. In this study, we have investigated the molecular basis of colistin resistance in 13 MDR K. pneumoniae strains isolated from 12 patients in a teaching hospital in Sousse, Tunisia. Whole-genome sequencing (WGS) was used to decipher the molecular mechanism of colistin resistance and to identify the resistome of these CoRKp isolates. It revealed a genome of ca. 5.5 Mbp in size with a G+C content of 57%, corresponding to that commonly observed for K. pneumoniae These isolates belonged to the 5 different sequence types (ST11, ST15, ST101, ST147, and ST392), and their resistome was composed of acquired ß-lactamases, including extended-spectrum beta-lactamase and carbapenemase genes (blaCTX-M-15, blaOXA-204, blaOXA-48, and blaNDM-1 genes), aminoglycoside resistance genes [aac(6')Ib-cr, aph(3″)-Ib, aph(6)-Id, and aac(3)-IIa], and fosfomycin (fosA), fluoroquinolone (qnr-like), chloramphenicol, trimethoprim, and tetracycline resistance genes. All of the isolates were identified as having a mutated mgrB gene. Mapping reads with reference sequences of the most common genes involved in colistin resistance revealed several modifications in mgrB, pmr, and pho operons (deletions, insertions, and substitutions) likely affecting the function of these proteins. It is worth noting that among the 12 patients, 10 were treated with colistin before the isolation of CoRKp No plasmid encoding mcr-1 to mcr-5 genes was found in these isolates. This study corresponds to the first molecular characterization of a collection of CoRKp strains in Tunisia and highlights that the small-transmembrane protein MgrB is a main mechanism for colistin resistance in K. pneumoniae.

Outbreak of colistin-resistant carbapenemase-producing Klebsiella pneumoniae in Tunisia.

OBJECTIVES: Mechanisms of colistin and carbapenem resistance among a collection of Klebsiella pneumoniae isolates recovered in a university hospital in Tunisia were studied. METHODS: In vitro antimicrobial susceptibility testing, S1 nuclease pulsed-field gel electrophoresis (S1-PFGE), Southern blotting and PCR-based replicon typing (PBRT) were performed. Extended-spectrum ß-lactamases (ESBLs), carbapenemases, AmpC-type enzymes and mgrB genes were detected by PCR and sequencing. Clonality of isolates was assessed by PFGE and multilocus sequence typing (MLST). RESULTS: Of 940 Enterobacteriaceae isolates recovered from June 2015 to March 2016 in Tahar Sfar Hospital (Mahdia, Tunisia), 220 were identified as K. pneumoniae, among which 29 were carbapenem-resistant. Carbapenem resistance was mostly due to expression of blaOXA-48 or blaOXA-204 in combination with blaCMY-4. Seven isolates carried blaNDM-1, of which two also harboured blaOXA-48, together with blaCMY-16 in one of them. All but two isolates also harboured blaCTX-M-15. All 20 blaOXA-48 genes were part of transposon Tn1999 on an IncL plasmid, whereas blaOXA-204 was found on transposon Tn2016 on an IncA/C plasmid. Finally, all blaNDM-1 genes were located within a Tn125 transposon on an IncFIIk plasmid. Interestingly, 7 (24.1%) of 29 carbapenem-resistant isolates were resistant to colistin, of which 6 were assigned to ST101, had similar PFGE profiles and presented the same 2-kb insertion in the mgrB gene. CONCLUSIONS: This study reports, for the first time in Tunisia, the full molecular characterisation of colistin resistance in K. pneumoniae. There is an urgent need for control measures and prudent use of colistin in treatment of infections with carbapenemase-producing K. pneumoniae.

Extended-spectrum ß-lactamases and plasmid-mediated quinolone resistance in enterobacterial clinical isolates from neonates in Tunisia.

This study was conducted to investigate extended-spectrum-ß-lactamase (ESBL) producing Enterobacteriaceae isolates from the Center of Maternity and Neonatology of Monastir, Tunisia. Fourty-six strains out of 283 were found to produce ESBL: Klebsiella pneumoniae (n = 37), Escherichia coli (n = 6), Enterobacter cloacae (n = 2), and Citrobacter freundi (n = 1). Genotyping analysis, using ERIC2 and RAPD, showed that strains were clonally unrelated. PCR amplification followed by sequencing revealed that all strains produced CTX-M-15. This enzyme was co-produced with TEM and SHV determinants in 34 and 36 strains respectively. The blaCTXM-15 gene was bracked by ISEcp1 and/or IS26 in 42 out of the 46 ESBL positive strains. The quinolone resistance determinants were associated to the ESBL producing isolates: we identified the qnrB1 gene in six isolates and the aac(6')-Ib-cr gene in five isolates. This epidemiological study shows the widespread of CTX-M-15 and qnr determinants among enterobacterial isolates from neonates hospitalized at the center of Maternity and Neonatology of Monastir suggesting either mother portage or horizontal transmission.

Phylogenetic analysis of partial VP7 gene of the emerging human group A rotavirus G12 strains circulating in Tunisia.

PURPOSE: Group A rotavirus (RVA) is the leading cause of severe gastroenteritis in children younger than 5 years. The most common human G-types are G1-4 and G9. G12 genotype is currently emerging worldwide, becoming the sixth most prevalent RVA G-genotype. In Tunisia, an emergence of G12 RVA strains was observed. To understand the evolution and origin of these Tunisian G12 strains, phylogenetic analyses were conducted. METHODOLOGY: A total of 1127 faecal samples were collected from Tunisian children under 5 years consulting for gastroenteritis between 2009 and 2014. Samples were screened by ELISA for the presence of RVA antigen. RVA-positive samples were used for the detection of G12 RVA strains by semi-nested RT-PCR. G12-positive specimens were subjected to VP4 genotyping reaction. PCR products of the G12-positive samples were sequenced and characterized by phylogenetic analysis of partial VP7 gene sequence. RESULTS: Globally, 270 (24 %) stool specimens were RVA-positive. Fourteen presented the G12 genotype (5.2 %) and were found to be in combination with either the P[6] (50.0 %) or the P[8] (50.0 %) genotype. Phylogenetic analysis revealed that all characterized Tunisian G12 strains clustered in the modern G12 lineage III and appear to form three different subclusters. CONCLUSION: Thus, the Tunisian G12 strains may have originated from not a single, but at least three distinct ancestral G12 strains. Detailed molecular characterization of the entire genome of these strains remains essential to help determine the extent of genetic variation and the relatedness of Tunisian G12 RVA strains to G12 strains described worldwide.

Distribution of rotavirus VP7 and VP4 genotypes circulating in Tunisia from 2009 to 2014: Emergence of the genotype G12.

Group A rotavirus (RVA) represents the most important aetiological agent of diarrhoea in children worldwide. From January 2009 to December 2014, a multi-centre study realized through 11 Tunisian cities was undertaken among children aged <5 years consulting or hospitalized for acute gastroenteritis. A total of 1127 faecal samples were collected. All samples were screened by ELISA for the presence of RVA antigen. RVA-positive samples were further analyzed by PAGE and used for G/P-genotyping by semi-nested multiplex RT-PCR. Globally, 270 specimens (24 %) were RVA-positive, with peaks observed annually between November and March. Nine different electropherotypes could be visualized by PAGE, six with a long profile (173 cases) and two with a short one (seven cases). Mixed profiles were detected in two cases. Among the 267 VP7 genotyped strains, the predominant G- genotype was G1 (39.6 %) followed by G3 (22.2 %), G4 (13 %), G9 (11.5 %), G2 (5.2 %) and G12 (5.2 %). Among the 260 VP4 genotyped strains, P[8] genotype was the predominant (74.5 %) followed by P[6] (10.4 %) and P[4] (5.5 %). A total of 257 strains (95.2 %) could be successfully G- and P-genotyped. G1P[8] was the most prevalent combination (34.4 %), followed by G3P[8] (16.3 %), G9P[8] (10.3 %), G4P[8] (8.9 %), G2P[4] (4 %), G12P[6] (2.6 %) and G12P[8] (1.9 %). Uncommon G/Pgenotype combinations, mixed infections and untypeable strains were also detected. This is the first report, in Tunisia, of multiple detection of an emerging human RVA strain, G12 genotype. This study highlighted the need for maintaining active surveillance of emerging strains in Northern Africa.

Impact of food animal trade on the spread of mcr-1-mediated colistin resistance, Tunisia, July 2015.

We report a high prevalence of MCR-1 and CTX-M-1-producing Escherichia coli in three Tunisian chicken farms. Chickens were imported from France or derived from French imported chicks. The same IncHI2-type plasmid reported to carry those genes in cattle in France and in a food sample in Portugal was found in Tunisian chickens of French origin. This suggests a significant impact of food animal trade on the spread of mcr-1-mediated colistin resistance in Europe.

[Association between bacterial resistance and antimicrobial consumption]. / Relations entre la résistance bactérienne et la consommation des antibiotiques.

UNLABELLED: The evolution of multidrug resistance remains an alarming topic due to selection pressure related to the inappropriate use of antibiotics. OBJECTIVES: Our work is in this perspective and focuses on the evolution of the consumption of antibiotics active on gram-negative bacilli, and the evolution of bacterial resistance. MATERIALS AND METHODS: International indicator of antibiotic consumption was based on the method of Defined Daily Dose reported to the number of days of hospitalization. The search for a correlation between bacterial resistance and antibiotic consumption was conducted by the Spearman test. RESULTS: A statistically significant correlation was identified between the rates of enterobacteriaceae resistant to 3(rd) generation cephalosporin, particularly those secreting beta-lactamases with extended spectrum, and consumption of 3(rd) generation cephalosporin (p= 0.002) and imipenem (p= 0.04). Also, a statistically significant relationship between the multi-resistant bacteria and the rate of consumption of colistin (p= 0.041) and fluoroquinolones (p= 0.002) was also reported in this study. CONCLUSION: Monitoring of both evolution of multidrug resistance and the use of antibiotics helps us to better understand the situation and establish more efficient antibiotic protocols.