Objectives CTX-M extended-spectrum β-lactamases (ESBLs) are emerging worldwide. Fast and reliable detection techniques may become mandatory for implementing proper treatment and infection control ...measures. Here, a blaCTX-M-specific LightCycler real-time PCR (LC-PCR) assay based on hybridization probes was developed. Methods Urine samples positive for Gram-negative bacilli as revealed by Gram staining were collected over a 3 month period at Bicêtre hospital, France. Aliquots of these urine samples were frozen for subsequent molecular analysis, and the bacteria were cultured and identified by standard bacteriological techniques (biochemical tests, disc diffusion antibiogram and synergy testing). LC-PCR and standard PCR followed by sequencing was performed on all ESBL-positive and on 70 randomly chosen ESBL-negative urine samples. Results Over the study period, 810 urine samples were collected from 655 patients. Thirty-six ESBL-producing Enterobacteriaceae, mostly Escherichia coli (77%), were identified from 29 patients, of which half were outpatients. Twenty-five urine samples (19 patients) were found to be positive for blaCTX-M genes using the LC-PCR assay. The blaCTX-M genes belonged to the blaCTX-M-1, blaCTX-M-9 and blaCTX-M-2 groups (68%, 24% and 8%, respectively). Standard PCR and sequencing of the entire blaCTX-M genes confirmed the LC-PCR results; 17 CTX-M-15, 6 CTX-M-9 and 2 CTX-M-2. Among the remaining ESBLs, eight were of the TEM type and three of the SHV type. Conclusions The LC-PCR assay represents a powerful tool for rapid identification of CTX-M producers in urine samples.
•A. baumannii isolates producing GES-11 ESBL and OXA-23 carbapenemase from Tunisia as early as 2006.•We illustrate the emergence of plasmid-encoded GES-11 in A. baumannii in North Africa.•We ...illustrate the transferability of these plasmid-encoded GES-type β-lactamases in A. baumannii.•Carbapenem-resistant A. baumannii in North African hospitals is a matter of concern.
Acinetobacter baumannii is an emerging threat in healthcare facilities owing to its ability to be multidrug-resistant (MDR) and to be involved in outbreaks. GES-type extended-spectrum β-lactamases (ESBLs) have been increasingly identified in A. baumannii. In this study, clinical A. baumannii isolates were characterised using standard biochemical methods and antibiotic susceptibility testing. Antibiotic resistance genes were sought by PCR and sequencing. Genetic support was characterised using S1 nuclease pulsed-field gel electrophoresis (PFGE) mapping, conjugation and electroporation assays. The genetic environment was investigated by PCR, and genetic relatedness was investigated by PFGE. Two MDR A. baumannii clinical isolates susceptible only to colistin and rifampicin were isolated from a tracheal aspirate of a 49-year-old woman hospitalised in 2006 at the Military Hospital of Tunis, Tunisia, and from a tracheal aspirate of a 53-year-old man hospitalised in 2010 at the Institut Orthopédique Mohamed El Kassab of Tunis, Tunisia. PCR revealed that the two isolates harboured the acquired carbapenemase blaOXA-23 and ESBL blaGES-11 genes along with chromosomally-encoded blaOXA-51 and blaADC-like genes. PFGE revealed that these A. baumannii isolates were unrelated; nevertheless, plasmid analysis revealed a similar sized plasmid following electrophoresis of the isolates. In addition, A. baumannii CIP70.10 transformants displayed similar resistance patterns. blaGES-11 was integron-borne and the ISAbaI element was identified upstream of blaOXA-23 and blaADC-like. Here we described two unrelated clinical A. baumannii isolates producing GES-11 ESBL and OXA-23 carbapenemase from two Tunisian hospitals. This work further illustrates the emergence of GES-type β-lactamases in A. baumannii in North Africa as early as 2006.
Retrotransposition of LINEs and other retroelements increases repetition in mammalian genomes and can cause deleterious mutations. Recent insertions of two full‐length L1s, L1spa and L1Orl, caused ...the disease phenotypes of the spastic and Orleans reeler mice respectively. Here we show that these two recently retrotransposed L1s are nearly identical in sequence, have two open reading frames and belong to a novel subfamily related to the ancient F subfamily. We have named this new subfamily TF (for transposable) and show that many full‐length members of this family are present in the mouse genome. The TF 5′ untranslated region has promoter activity, and TF‐type RNA is abundant in cytoplasmic ribonucleoprotein particles, which are likely intermediates in retrotransposition. Both L1spa and L1Orl have reverse transcriptase activity in a yeast‐based assay and retrotranspose at high frequency in cultured cells. Together, our data indicate that the TF subfamily of L1s contains a major class of mobile elements that is expanding in the mouse genome.
This study was conducted to investigate the presence of Enterococcus spp. in meat, meat products and seafood. A hundred and four samples were randomly collected from different geographic localities ...in Libya. The samples were subjected to microbiological analysis for enumeration and isolation of Enterococcus spp. by conventional cultural and molecular identification using PCR and partial sequencing of 16S rDNA techniques. Out of 104 samples, 73 (70.2%) isolates were found to be enterococci based on their cultural characteristics on ESD medium. However, out of 36 samples subjected to molecular identification, only six isolates were confirmed to be Enterococcus spp. using PCR and partial sequencing of 16S rDNA technique. All enterococci strains tested for their antibiotic sensitivity profiles showed high percentage of multi-resistance phenotype. These results can be used for further studies on enterococci as an emerging food borne pathogen and its role in human infection in Libya and would suggest that meat, meat products and seafood might play a role in the spreading of enterococci through the food chain with antimicrobial resistance characteristics.
We previously isolated two human L1 elements (L1.2 and LRE2) as the progenitors of disease-producing insertions. Here, we show these elements can actively retrotranspose in cultured mammalian cells. ...When stably expressed from an episome in HeLa cells, both elements retrotransposed into a variety of chromosomal locations at a high frequency. The retrotransposed products resembled endogenous L1 insertions, since they were variably 5′ truncated, ended in poly(A) tracts, and were flanked by target-site duplications or short deletions. Point mutations in conserved domains of the L1.2-encoded proteins reduced retrotransposition by 100- to 1000-fold. Remarkably, L1.2 also retrotransposed in a mouse cell line, suggesting a potential role for L1-based vectors in random insertional mutagenesis.
Carbapenem-resistant Acinetobacter baumannii isolates producing carbapenem-hydrolysing oxacillinases are emerging worldwide. These enzymes are divided into four phylogenetic subgroups: OXA-23-like, ...OXA-51-like, OXA-24-like and OXA-58-like. A PCR-based approach linked to pyrosequencing analysis was developed to identify the genes for these β-lactamases. Carbapenem-hydrolysing oxacillinases were rapidly and unambiguously identified in a collection of carbapenem-resistant clinical isolates of A. baumannii and Acinetobacter junii. Pyrosequencing may provide a rapid tool for identification of OXA variants, thus avoiding delays inherent in classical sequencing methods.
Flavobacterium johnsoniae CIP100931 is resistant to most β-lactam antibiotics and has a decreased susceptibility to carbapenems. A β-lactamase gene was cloned and expressed in Escherichia coli DH10B. ...The purified β-lactamase, JOHN-1, with a pI value of 9.0 and with a determined relative molecular mass of ∼27 kDa was found to be a monomeric zinc-dependent enzyme that hydrolyses penicillins, narrow- and expanded-spectrum cephalosporins, carbapenems, but not monobactams. Sequence analysis revealed that JOHN-1 is a molecular class B β-lactamase that is most closely related to BlaB from Chryseobacterium meningosepticum and IND-1 from Chryseobacterium indologenes (47% and 41% amino acid identity, respectively). JOHN-1 is a new member of the highly divergent subclass B1 lineage of metallo-enzymes. Although F. johnsoniae and Chryseobacterium spp. are phylogenetically related bacteria, this report further underlines the heterogeneity of class B β-lactamases that are naturally produced by environmental Gram-negative aerobes and that are now recognized as the most important reservoir for these β-lactamase genes.
The genus
Vibrio
includes several food-borne pathogens that cause a spectrum of clinical conditions including septicemia, cholera and milder forms of gastroenteritis. Several
Vibrio
spp. are commonly ...associated with food-borne transmission including
Vibrio cholerae
,
Vibrio parahemolyticus
, and
Vibrio vulnificus
. Microbiological analysis for enumeration and isolation of
Vibrio
spp. were carried out for a total of 93 samples of seafood, meat and meat products from different geographic localities in Libya (Tripoli, Regdalin, Janzour and Tobruk).
Vibrio
spp. were detected by conventional cultural and molecular method using PCR and sequencing of 16S rDNA. Out of the 93 cultured samples only 48 (51.6%) yielded colonies on Thiosulfate Citrate Bile Salt agar (TCBS) with culture characteristics of
Vibrio
spp. More than half (n=27) of processed seafood samples (n=46) yielded colonies on TCBS, while only 44.6 % of samples of meat and meat products showed colonies on TCBS. Among cultured seafood samples, the highest bacterial count was recorded in clam with a count of 3.8 ×10
4
CFU\g. Chicken burger samples showed the highest bacterial count with 6.5 ×10
4
CFU\g. Molecular analysis of the isolates obtained in this study, showed that 11 samples out of 48 (22.9%) were
Vibrio
spp.
Vibrio parahemolyticus
was isolated from camel meat for the first time. This study is an initial step to provide a baseline for future molecular research targeting
Vibrio
spp. foodborne illnesses. This data will be used to provide information on the magnitude of such pathogens in Libyan seafood, meat and meat products.
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