Enterobacter cloacae Complex and CTX-M Extended-Spectrum ß-Lactamases in Algeria.

We evaluated the ß-lactam resistance phenotypes of clinical and environmental strains of the Enterobacter cloacae complex (ECC) isolated from three Algerian hospitals. The first combination of API 20E, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS), and hsp60 genetic clustering methodologies were carried out for the identification of ECC strains. Our research showed that API 20E and MALDI TOF MS are satisfactory in genus identification of ECC strains, but sequence-based methods are then necessary to discriminate the species and subspecies levels. Among 36 ECC strains, 94.44% belonged to Enterobacter hormaechei species. Twenty-five isolates clustered with the reference strain of E. hormaechei subsp. xiangfangensis, making it the most frequently isolated subspecies. Enterobacter kobei was found only once (2.77%). All ECC isolates were phenotypically extended-spectrum ß-lactamase (ESBL) producers and were resistant to ticarcillin, piperacillin, cefoxitin, cefotaxime, ceftazidime, ceftriaxone, and aztreonam, but susceptible to ertapenem and imipenem. The genetic analyses only allowed the detection of resistance genes of the CTX-M-1 group (32 strains, 88.9%), including CTX-M-15 (30 strains), CTX-M-3 (1 strain), and CTX-M-22 (1 strain). We report for the first time the detection of CTX-M-22 among ECC strains in an Algerian hospital (Tlemcen hospital). None of the isolated strains harbored CTX-M-2, CTX-M-9, or CTX-M-8/25 group genes. In this review, we address recent comparison in the identification methods of multidrug-resistant E. cloacae complex in Algeria, focusing also on the CTX-M ESBLs. This represents a serious public health challenge, which requires the clarification of the current situation and warrants the reinforcement of hygiene measures in the Algerian hospitals.

Occurence of ArmA and RmtB Aminoglycoside Resistance 16S rRNA Methylases in Extended-Spectrum ß-Lactamases Producing Escherichia coli in Algerian Hospitals.

The aim of this study, was to characterize the extended-spectrum-ß-lactamases (ESBLs) producing clinical strains of Escherichia coli isolated between January 2009 and June 2012 from Algerian hospitals and to determine the prevalence of 16S rRNA methylase among them. Sixty-seven ESBL-producers were detected among the 239 isolates included: 52 CTX-M-15-producers, 5 CTX-M-3-producers, 5 CTX-M-1-producers, 2 CTX-M-14-producers, 2 SHV-12-producers and one TEM-167-producer. Among the ESBL-producing strains twelve harbored 16S rRNA methylase genes: 8 rmtB and 4 armA. rmtB was located on a IncFIA plasmid and armA was located either on a IncL/M or a IncFIA plasmid. RmtB-producing isolates were genotypically related and belonged to the sequence type ST 405 whereas ArmA-producing isolates belonged to ST10, ST 167, and ST 117. This first description of 16S rRNA methylases among E. coli in Algerian hospitals pointed out the necessity to establish control measures to avoid their dissemination.

Molecular epidemiology of carbapenem-resistant Pseudomonas aeruginosa clinical strains isolated from western Algeria between 2009 and 2012.

Infections caused by carbapenem-resistant Pseudomonas aeruginosa strains represent a major therapeutic and epidemiological problem. The aim of this study was to characterize carbapenem resistance in 89 clinical strains of P. aeruginosa isolated from three hospitals in western Algeria between October 2009 and November 2012. Minimum inhibitory concentrations (MICs) of imipenem were determined by the Etest method. Screening for metallo-ß-lactamase (MßL) was performed using Etest MßL strips, and a PCR was conducted to detect carbapenemase-encoding genes. The amplification of the oprD gene followed by a sequencing reaction was performed for all strains resistant to imipenem. The clonality of 53 P. aeruginosa strains was demonstrated using multilocus sequence typing (MLST). Among the 89 isolates, 35 (39.33%) were found to be resistant to IMP (MICs ≥16 µg/ml). The blaVIM-2 gene was detected in two strains. The remaining imipenem-resistant isolates showed the presence of oprD mutations. The MLST analysis differentiated strains into various clones and the strains from the same clone had an identical sequence of the oprD gene. We report the second detection in 2010 of blaVIM-2 in Algerian P. aeruginosa strains. We also found that oprD mutations were the major determinant of high-level imipenem resistance. We demonstrate that these oprD mutations can be used as a tool to study the clonality in P. aeruginosa isolates.

Prevalence of carbapenemase-encoding genes including New Delhi metallo-ß-lactamase in Acinetobacter species, Algeria.

BACKGROUND: Nosocomial infections caused by carbapenem-resistant Acinetobacter spp are a global health problem. The aim of this study was to investigate the molecular epidemiology and the genetic support of carbapenem resistance in Acinetobacter spp clinical isolates recovered from three different hospitals in western Algeria from 2008 to 2012. METHODS: A total of 113 Acinetobacter spp isolates were identified by matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometry. Antimicrobial susceptibility testing was carried out, and minimum inhibitory concentrations (MICs) were determined by the dilution method on Mueller-Hinton agar for ß-lactams, aminoglycosides, fluoroquinolones, and colistin. The characterization of ß-lactamases was investigated by phenotypic tests for the detection of metallo-ß-lactamases and oxacillinases. Resistance genes were screened for by quantitative PCR and sequenced when positive. RESULTS: Among the 113 isolates, 80 (70.8%) were found to be resistant to imipenem with MICs ranging from 64 to 512µg/ml. The blaOXA-23-like gene was detected in 50% (40/80) of the isolates and the blaOXA-24-like gene was detected in 21.2% (17/80) of the isolates. In addition, the metallo-ß-lactamase blaNDM-1-like was detected in five isolates (6.2%). CONCLUSIONS: This study represents the first description of autochthonous Acinetobacter spp producing metallo-ß-lactamase blaNDM-1-like and oxacillinases blaOXA-23-like and blaOXA-24-like in western Algeria.

Biotyping of multidrug-resistant Klebsiella pneumoniae clinical isolates from France and Algeria using MALDI-TOF MS.

BACKGROUND: Klebsiella pneumoniae is one of the most important pathogens responsible for nosocomial outbreaks worldwide. Epidemiological analyses are useful in determining the extent of an outbreak and in elucidating the sources and the spread of infections. The aim of this study was to investigate the epidemiological spread of K. pneumoniae strains using a MALDI-TOF MS approach. METHODS: Five hundred and thirty-five strains of K. pneumoniae were collected between January 2008 and March 2011 from hospitals in France and Algeria and were identified using MALDI-TOF. Antibiotic resistance patterns were investigated. Clinical and epidemiological data were recorded in an Excel file, including clustering obtained from the MSP dendrogram, and were analyzed using PASW Statistics software. RESULTS: Antibiotic susceptibility and phenotypic tests of the 535 isolates showed the presence of six resistance profiles distributed unequally between the two countries. The MSP dendrogram revealed five distinct clusters according to an arbitrary cut-off at the distance level of 500. Data mining analysis of the five clusters showed that K. pneumoniae strains isolated in Algerian hospitals were significantly associated with respiratory infections and the ESBL phenotype, whereas those from French hospitals were significantly associated with urinary tract infections and the wild-type phenotype. CONCLUSIONS: MALDI-TOF was found to be a promising tool to identify and differentiate between K. pneumoniae strains according to their phenotypic properties and their epidemiological distribution. This is the first time that MALDI-TOF has been used as a rapid tool for typing K. pneumoniae clinical isolates.

Hierarchical clustering as a rapid tool for surveillance of emerging antibiotic-resistance phenotypes in Klebsiella pneumoniae strains.

Antimicrobial resistance is on the rise, and its early detection and surveillance are critical to implement effective control measures. The aim of this study was to develop a rapid hierarchical clustering bioinformatic tool for application on antibiotic susceptibility testing (AST) results (resistant, intermediate, sensitive) of a series of Klebsiella pneumoniae clinical isolates from Algeria and from France for surveillance of antibiotic-resistance phenotypes. A total of 1011 K. pneumoniae strains were collected from August 2008 to December 2012: 221 clinical isolates from western Algeria and 790 clinical isolates from Marseille, France. AST against a panel of 16 antibiotics was done for all isolates. Results of AST were introduced into MultiExperiment Viewer (MeV) software to perform hierarchical clustering, with resistant, intermediate and sensitive being translated to 1, 0 and -1 values, respectively. Hierarchical clustering results were compared to standard resistance phenotypes to evaluate the accuracy of the method. Based on the AST results, the 221 K. pneumoniae strains from Algeria could be separated into six phenotype groups as regards their resistance to ß-lactam compounds: extended spectrum ß-lactamase (ESBL) (68.3 %), ESBL associated with cephalosporinase (13.1 %), cephalosporinase (0.9 %), penicillinase (3.6 %) and wild-type (14.0 %). Hierarchical clustering by the MeV software applied to the AST results for all 1011 isolates generated clusters that were significantly representative of phenotypic classification and geographical origin, in less than 1 min. Moreover, adding to the dataset the AST results of a K. pneumoniae NDM-1 positive strain, the only strain resistant to imipenem in the series, immediately generated a new branch in the dendrogram. We have developed a rapid and simple hierarchical clustering tool for application on AST results that was able to survey qualitatively and quantitatively the prevalence of known and unknown phenotypes. This tool could be easily implemented in routine clinical microbiology laboratories.

Molecular and epidemiological characterization of enterobacterial multidrug-resistant strains in Tlemcen Hospital (Algeria) (2008-2010).

Seventy-one isolates of Enterobacteriaceae (17 Escherichia coli, 50 Klebsiella pneumoniae, and 4 Enterobacter cloacae) producing extended-spectrum-ß-lactamases (ESBLs) were collected between April 2008 and March 2010 in an intensive care unit and surgical ward of Tlemcen Hospital (West of Algeria). Sequencing identified the bla(CTX-M-15) determinant in 69 isolates and bla(CTX-M-3) in 2 isolates. None of the studied strains produced the class D carbapenemase OXA-48. Repetitive Extragenic palindromic polymerase chain reaction showed a high degree of genotypic diversity among E. coli strains and two major clonal populations of K. pneumoniae (CKp1 n=11 and CKp5 n=25) which were further identified as members of the multilocus sequence typing types (ST931) and (ST15), respectively. The ST15 isolates harbored more resistance genes and virulence factors than the ST931 isolates. The characterization of the spacer region between ISEcp1 and bla(CTX-M) for CTX-M-15 producers individualized two populations. One that derived from the CTX-M-3 under Algerian clinical context and one that is universally found. The dissemination of ESBLs in the studied Enterobacteriaceae isolates was mainly due to the epidemic clones of K. pneumoniae and to genetic transit of plasmids among unrelated strains.

Rapid detection of carbapenem resistance in Acinetobacter baumannii using matrix-assisted laser desorption ionization-time of flight mass spectrometry.

Rapid detection of carbapenem-resistant Acinetobacter baumannii strains is critical and will benefit patient care by optimizing antibiotic therapies and preventing outbreaks. Herein we describe the development and successful application of a mass spectrometry profile generated by matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) that utilized the imipenem antibiotic for the detection of carbapenem resistance in a large series of A. baumannii clinical isolates from France and Algeria. A total of 106 A. baumannii strains including 63 well-characterized carbapenemase-producing and 43 non-carbapenemase-producing strains, as well as 43 control strains (7 carbapenem-resistant and 36 carbapenem-sensitive strains) were studied. After an incubation of bacteria with imipenem for up to 4 h, the mixture was centrifuged and the supernatant analyzed by MALDI-TOF MS. The presence and absence of peaks representing imipenem and its natural metabolite was analyzed. The result was interpreted as positive for carbapenemase production if the specific peak for imipenem at 300.0 m/z disappeared during the incubation time and if the peak of the natural metabolite at 254.0 m/z increased as measured by the area under the curves leading to a ratio between the peak for imipenem and its metabolite being <0.5. This assay, which was applied to the large series of A. baumannii clinical isolates, showed a sensitivity of 100.0% and a specificity of 100.0%. Our study is the first to demonstrate that this quick and simple assay can be used as a routine tool as a point-of-care method for the identification of A. baumannii carbapenemase-producers in an effort to prevent outbreaks and the spread of uncontrollable superbugs.