Molecular Characterization of Clinical Carbapenem-Resistant Enterobacteriaceae Isolates from Sétif, Algeria.

This study aimed to determine the incidence and the molecular mechanisms of carbapenem-resistant Enterobacteriaceae in patients from the Sétif University Hospital, Algeria. Nonduplicate clinical bacterial isolates recovered from patients attending the University Hospital of Sétif were collected between April and October 2018. Species identification was performed by MALDI-TOF/MS (matrix-assisted laser desorption ionization-time of flight mass spectrometry) method. The susceptibility of the isolates to carbapenems was determined using the disc diffusion method. The carbapenem resistant isolates were screened for the presence of common carbapenemase genes (blaKPC, blaOXA-48, blaVIM, blaIMP, and blaNDM) and extended-spectrum ß-lactamase (blaCTX, blaTEM, and blaSHV) using PCR and sequencing technique. A total of 123 nonrepetitive Enterobacteriaceae isolates were obtained. Klebsiella pneumoniae (n = 52/42.28%), Escherichia coli (n = 24/19.51%), and Enterobacter cloacae (n = 19/15.45%) were the most prevalent species. The Carba-NP test showed that 6 out of 123 isolates carried carbapenemase enzymes. OXA-48 was found in five isolates (four K. pneumoniae and one E. coli) and NDM-5 in one E. cloacae isolate. We reported for the first time in Algeria the presence of NDM-5 carbapenemase enzyme in a clinical E. cloacae isolate.

Chemical Composition and Antibacterial Activity of Essential Oils from the Algerian Endemic Origanum glandulosum Desf. against Multidrug-Resistant Uropathogenic E. coli Isolates.

Antibiotics are becoming ineffective against resistant bacteria. The use of essential oils (EOs) may constitute an alternative solution to fight against multidrug-resistant bacteria. This study aims to determine the chemical composition of EOs from five populations of the endemic Algerian Origanum glandulosum Desf. and to investigate their potential antibacterial activity against multidrug-resistant uropathogenic E. coli strains. The EOs were obtained by hydrodistillation and their composition was investigated by gas chromatography/mass spectrometry (GC/MS). The antibacterial activity was evaluated by the disc diffusion method against eight E. coli strains (six uropathogenic resistant and two referenced susceptible strains). Minimum inhibitory and bactericidal concentrations (MIC/MBC) were obtained by the broth microdilution method. The main EO components were thymol (15.2-56.4%), carvacrol (2.8-59.6%), γ-terpinene (9.9-21.8%) and p-cymene (8.5-13.9%). The antibacterial tests showed that all the EOs were active against all the strains, including the multidrug-resistant strains. The EO from the Bordj location, which contained the highest amount of carvacrol (59.6%), showed the highest antibacterial activity (inhibition diameters from 12 to 24.5 mm at a dilution of 1/10). To our knowledge, this is the first description of the activity of O. glandulosum EOs against resistant uropathogenic strains. Our study suggests that O. glandulosum EO could be used in some clinical situations to treat or prevent infections (e.g., urinary tract infections) with multidrug-resistant strains.

Development of real-time PCR assay allowed describing the first clinical Klebsiella pneumoniae isolate harboring plasmid-mediated colistin resistance mcr-8 gene in Algeria.

OBJECTIVES: We aimed to develop here a specific real-time PCR assay with TaqMan® probe to detect efficiently bacterial strains harboring the new plasmid mediated-colistin resistance mcr-8 gene. METHODS: Specific primers and probe for mcr-8 gene were designed from sequences alignment of all mcr genes variants. Specificity of the designed primers and probe were first checked par BlastN analysis and by in silico PCR. The analytical sensitivity and specificity tests were performed in vitro on a panel of 290 genomic DNA of Gram-negative bacteria and 250 metagenomic DNA from human stool samples. Whole genome sequencing (WGS) was performed here using MiSeq technology. RESULTS: Designed primers and probe were 100% specific tomcr-8 gene by BlastN and in silico PCR analysis. Real-time PCR screening of a collection of clinical isolates resulted to one positive Klebsiella pneumoniae isolate (KP95). WGS confirmed that this isolate harbored the mcr-8 gene and other resistance genes such as blaOXA-48, blaCTX-M-15 ß-lactamases. Our real-time PCR was highly sensitive on a 10-fold dilution serie from a calibrated inoculum at 108 CFU/mL with a limit of detection at 55 CFU/mL. CONCLUSION: To the best of our knowledge, we propose here, the first real-time PCR assay targeting mcr-8 gene with high specificity and sensitivity, able to detect mcr-8 gene in less than 2 h from any DNA sample. This real-time PCR assay allowed the first description of a clinical K. pneumoniae strain harboring the mcr-8 gene in Algeria.

High Prevalence of Multidrug-Resistant Escherichia coli in Urine Samples from Inpatients and Outpatients at a Tertiary Care Hospital in Sétif, Algeria.

The worldwide dissemination of multidrug-resistant (MDR) Enterobacteriaceae is a major public health issue. The aim of this study was to investigate the prevalence of MDR Escherichia coli (MDR-EC) isolates, in inpatients/outpatients with urinary tract infections at Sétif University Hospital (Algeria). Bacterial cultures were obtained from 426 of the 3,944 urine samples collected from January 2015 to February 2017. Among these cultures, 215 E. coli isolates were identified by mass spectrometry, and 38 (17.7%) were MDR-EC (disk diffusion method): 36 produced only extended-spectrum ß-lactamases (ESBL), one ESBL and a carbapenemase, and one only a cephalosporinase (double-disk synergy test). Multiplex PCR and sequencing analyses showed that 37 ESBL-producing isolates harbored genes encoding CTX-M enzymes (CTX-M-15 in 33 isolates, 89.19%; and CTX-M-14 group in four isolates, 10.81%). One CTX-M-15-producing isolate co-expressed also an OXA-48-like carbapenemase. Phylogenetic group analysis of the 37 ESBL-producing and 178 non-ESBL-producing isolates indicated that the most common phylogenetic group was B2 (54.05% of ESBL-producing and 48.31% of non-ESBL-producing isolates), followed by A and D for ESBL-, and by B1, A, and F for non-ESBL-producing isolates. This is the first report highlighting the presence of MDR-EC isolates that produce both CTX-M and OXA-48-like enzymes in Sétif, Algeria.

Occurrence and clonal diversity of multidrug-resistant Klebsiella pneumoniae recovered from inanimate surfaces in Algerian hospital environment: First report of armA, qnrB and aac(6')-Ib-cr genes.

PURPOSE: The aim of this study is to characterize the molecular support of antibiotic resistance in MDR Klebsiella pneumoniae recovered from inanimate surfaces between March 2012 to February 2014 in three teaching hospitals (Setif, Bejaia and Constantine) in Algeria. RESULTS: Forty-four K. pneumoniae producing ESBL were detected and blaCTX-M-15 and blaCTX-M-3 were detected respectively in 41 and 3 isolates. These K. pneumoniae isolates producing ESBL were also resistant to gentamicin (87%), tobramicin (87%), ciprofloxacin (66%) and ofloxacin (62%). Aminoglycosides resistance genes detected were 16S rRNA methylase (armA), aminoglycoside acetyl-transferase (aac(6')-Ib), aminoglycoside nucleotidyl-transferase (aadA2) and aminoglycoside, phosphoryl-transferase (ant3″Ih-aac(6')-IId). Plasmid-mediated quinolone resistance (PMQR) genes detected were aac(6')-Ib-cr (34 isolates) and qnrB genes in (34 isolates). Multilocus sequence typing (MLST) resulted in 12 different sequence types (STs) regrouped into 5 clonal complexes (CC147, CC17, CC37, CC2 and CC23), one clonal group (CG485) and 4 singletons (ST1426, ST405, ST1308, ST873). CONCLUSION: Here, we report the detection of the ESBLs encoding gene linked with plasmid-mediated quinolone resistance (PMQR) and aminoglycosides resistance recovered from inanimate surfaces in hospital environment.

Hemodialysis catheter-related infection: rates, risk factors and pathogens.

The main complication of central venous catheter (CVC) in hemodialysis is infection. Identifying CVC related infection (CVC-RI) risk factors and causative micro-organisms is important for setting prevention policies. There were no data regarding CVC-RI in hemodialysis in Algeria. To determine rates of CVC-RI in hemodialysis in Setif university hospital, risk factors and causative microorganisms, we conducted a prospective study from November 2014 to May 2015 involving patients with CVC in hemodialysis. Micro-organisms isolated from semi quantitative culture of CVC and blood culture were identified and tested for antibiotic susceptibility using the automated MicroScan system (DADE Behring, Sacramento, CA, USA). Chi-square test was performed to compare demographic and clinical variables (age, sex, comorbidities, duration of CVC, insertion site) in the groups of patients with and without CVC-RI. P<0.05 was considered statistically significant. All analyses were performed using SPSS V17 for Windows statistical package (SPSS Inc., Chicago, IL, USA). 94 patients and 152 CVC procedures were analyzed. 34 CVC-RI were documented with an incidence of 16.6 per 1000 CVC-days. Incidence of CVC related bloodstream infection (CVC-RBI) was 10.8 per 1000 CVC-days. Independent risk factors associated with CVC-RI were diabetes (P=0.01) and duration of catheterization (P= 0.01). Causative micro-organisms were: Klebsiella pneumoniae 26.5%, coagulase-negative staphylococci 23.5% and Staphylococcus aureus 23.5%. Micro-organisms were multidrug-resistant (MDR). Mortality was statistically associated to inadequate antibiotic therapy. The duration of CVC should be reduced by creation of fistulas. More compliance to hygiene measure is needed for decreasing CVC-RI and resistance rate.

Chemical analysis, antimicrobial and anti-oxidative properties of Daucus gracilis essential oil and its mechanism of action

Objective: To evaluate the essential oils (EO) composition, antimicrobial and antioxi-dant power of a local plant, Daucus gracilis (D. gracilis). Methods: The aerial parts of D. gracilis were subjected to hydro distillation by a Cle-venger apparatus type to obtain the EO which had been analyzed by gas chromatography and gas chromatography coupled with mass spectrometry, and screened for antimicrobial activity against five bacteria and three fungi by agar diffusion method. The mechanism of action of the EO was determined on the susceptible strains by both of time kill assay and lysis experience. The minimal inhibitory concentrations were determined by agar macro-dilution and micro-dilution methods. Anti-oxidative properties of the EO were also studied by free diphenyl-2-picrylhydrazyl radical scavenging and reducing power techniques. Results: The EO yielded 0.68 (v/w). The chemical analysis presented two dominant constituents which were the elemicin (35.3%) and the geranyl acetate (26.8%). D. gracilis EO inhibited the growth of Bacillus cereus and Proteus mirabilis significantly with minimal inhibitory concentrations of 17.15 mg/mL by the agar dilution method and 57.05 mg/mL and 114.1 mg/mL, respectively by liquid micro-dilution. A remarkable decrease in a survival rate as well as in the absorbance in 260 nm was recorded, which suggested that the cytoplasm membrane was one of the targets of the EO. The EO showed, also, important anti-oxidative effects with an IC50 of 0.002 mg/mL and a dose-dependent reducing power. Conclusions: D. gracilis EO showed potent antimicrobial and anti-oxidative activities and had acted on the cytoplasm membrane. These activities could be exploited in the food industry for food preservation.

First report of 16S rRNA methylase ArmA-producing Acinetobacter baumannii and rapid spread of metallo-ß-lactamase NDM-1 in Algerian hospitals.

PURPOSE: The aim of the present study was to characterize the molecular support of resistance to carbapenems, aminoglycosides and fluoroquinolones in carbapenem-resistant Acinetobacter baumannii clinical isolates recovered between January 2011 and April 2013 from Algerian hospitals. METHODS: Antibiotic susceptibility testing was performed using disk diffusion and Etest methods. Carbapenemase activity was detected using both MALDI-TOF mass spectrometry assay and via microbiological tests. Carbapenem, aminoglycoside and fluoroquinolone resistance determinants were studied by PCR and sequencing. Clonal relationships between strains were determined using Multi Locus Sequence Typing (MLST). RESULTS: A total of 47 imipenem-resistant A. baumannii were isolated and identified by MALDI-TOF mass spectrometry. All imipenem-resistant strains were positive in the modified Hodge test, and EDTA inhibited the activity of metallo-ß-lactamases enzymes in 11 strains. The blaOXA-23 gene was detected in 33 strains and the blaOXA-24 gene in 10 strains. The metallo-ß-lactamase blaNDM-1 gene was detected in 11 isolates (23.4%) from Algiers and Sétif, including 7 that co-expressed a blaOXA-23 gene. Resistance to aminoglycosides was due to the production of aminoglycoside-modifying enzymes, AAC(3)-Ia, AADA, ANT(2″)-I, APH(3')-VI, and 16S rRNA methylases, ArmA. The fluoroquinolone resistance was mainly associated with mutations at Ser83Leu and Ser80Leu of the gyrA and parC genes, respectively. MLST revealed five sequence types (STs), 1, 2, 19, 25, and 85. The imipenem-resistant A. baumannii ST2 was the predominant clone (35/47). CONCLUSIONS: Here we report for the first time clinical multidrug-resistant A. baumannii isolates harboring 16S rRNA methylase gene, armA, and rapid spread of metallo-ß-lactamase NDM-1 isolated from patients in Algeria.

Antibiotic resistance determinants of multidrug-resistant Acinetobacter baumannii clinical isolates in Algeria.

Antibiotic susceptibility testing was performed on 71 Acinetobacter baumannii clinical isolates, and presence of antibiotic resistance genes was screened for by PCR amplification and sequencing. Resistance rates were very high for aminoglycosides (22-80%), fluoroquinolones (>90%), and cephalosporins (>90%) but remained low for rifampin (2.8%) or null for colistin. Antibiotic resistance encoding genes detected were as follows: blaTEM-128 gene (74.6%), aph(3')-VI (50.7 %), aadA (63.4%), ant(2″)-I (14.1%), aac(3)-Ia (91.1%), aac(6')-Ib (4.2%), mutation Ser83Leu in gyrA (94.4%), double mutations Ser83Leu and Ser80Leu (or Ser84Leu) in gyrA and parC (69.0%), and mutation I581N in RRDR of the rpoB gene.