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.

Mortality among Patients with Nosocomial Infections in Tertiary Intensive Care Units of Sahloul Hospital, Sousse, Tunisia.

BACKGROUND: Nosocomial infections are public health issues that are associated with high mortality in intensive care units. This study aimed to determine nosocomial infection-associated mortality in Tunisian intensive care units and identify its risk factors. METHODS: A prospective cohort study was carried out in intensive care units of a Tunisian University Hospital. The ICUs-wide active surveillance of nosocomial infections has been performed between 1 July 2010 and 30 June 2011. Data collection was based on Rea-Raisin protocol 2009 of "Institut National de Veille Sanitaire" (InVS, Saint Maurice - France). We used Kaplan Meier survival analysis and Cox Proportional Hazard regression to identify independent risk factors of nosocomial infection-associated mortality. RESULTS: Sixty-seven patients presented nosocomial infection in the end of the surveillance. The mean age of patients was 44.71 ± 21.2 years. Of them, 67.2% were male and 32.8% female. Nosocomial bacteremia was the most frequent infection (68.6%). Nosocomial infection-associated mortality rate was 35.8% (24/67). Bacteremia (Hazard Ratio (HR)) = 3.03, 95% Confidential Interval (95% CI): [1.23 - 7.45], P = 0.016) and trauma (HR = 3.6, 95% CI: [1.16 - 11.2], P = 0.026) were identified by Cox regression as independent risk factors for NI-associated mortality. CONCLUSIONS: Our rate was relatively high. We need to improve the care of trauma patients and intensify the fight against nosocomial infections especially bacteremia.

Changes in EEG activity before and after exhaustive exercise in sedentary women in neutral and hot environments.

This study examined the effect of hyperthermia on brain electrical activity measured with encephalography during prolonged exhaustive exercise in a group of sedentary women (VO(2)max = 35 +/- 4 mL kg min(-1)). Two strenuous cycling exercises were performed either in neutral (N-Ex) or in heat (H-Ex) conditions. Tympanic temperature (Tty), heart rate (HR), body mass loss (BML), plasma volume decrease, and brain electrical activity [EEG: alpha (8-13 Hz) and beta(13-30 Hz)-band and alpha/beta index of fatigue: the ratio between EEG activity in the alpha band and beta-band] were recorded throughout the cycling sessions. The Tty increase 1.0 degrees C in the N-Ex and 1.8 degrees C in H-Ex. HR increased in both sessions but with significantly higher values during the H-Ex session when compared with the N-Ex session (p < 0.001) (from 85 +/- 4 beats min(-1) to 164 +/- 6 beats min(-1) and from 83 +/- 6 beats min(-1) to 181 +/- 8 beats min(-1), respectively in N-Ex and in H-Ex). This was associated with a significantly higher BML (p < 0.05) and a higher plasma volume decrease in the H-Ex session (p < 0.01). The alpha/beta index increased significantly during both trials particularly during the H-Ex session (p < 0.05). This was associated with a significant decrease of time to exhaustion (-34%). We suggest that exhausting work in the heat induced a change in gross brain activity (alpha/beta ratio) compared to a longer, less thermally demanding exposure. Fatigue in the heat could be attributed to central factors as well as thermal, cardiac and hydro-electrolytic impairment.