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1.  Proteomic Biomarkers Associated with Streptococcus agalactiae Invasive Genogroups 
PLoS ONE  2013;8(1):e54393.
Group B streptococcus (GBS, Streptococcus agalactiae) is a leading cause of meningitis and sepsis in newborns and an etiological agent of meningitis, endocarditis, osteoarticular and soft tissue infections in adults. GBS isolates are routinely clustered in serotypes and in genotypes. At present one GBS sequence type (i.e. ST17) is considered to be closely associated with bacterial invasiveness and novel proteomic biomarkers could make a valuable contribution to currently available GBS typing data. For that purpose we analyzed the protein profiles of 170 genotyped GBS isolates by Surface-Enhanced Laser Desorption/Ionization Time-of-Flight Mass Spectrometry (SELDI). Univariate statistical analysis of the SELDI profiles identified four protein biomarkers significantly discriminating ST17 isolates from those of the other sequence types. Two of these biomarkers (MW of 7878 Da and 12200 Da) were overexpressed and the other two (MW of 6258 Da and 10463 Da) were underexpressed in ST17. The four proteins were isolated by mass spectrometry-assisted purification and their tryptic peptides analyzed by LC-MS/MS. They were thereby identified as the small subunit of exodeoxyribonuclease VII, the 50S ribosomal protein L7/L12, a CsbD-like protein and thioredoxin, respectively. In conclusion, we identified four candidate biomarkers of ST17 by SELDI for high-throughput screening. These markers may serve as a basis for further studies on the pathophysiology of GBS infection, and for the development of novel vaccines.
doi:10.1371/journal.pone.0054393
PMCID: PMC3553121  PMID: 23372719
2.  Primary antibiotic resistance and associated mechanisms in Helicobacter pylori isolates from Senegalese patients 
Background
Antibiotic combination therapy for Helicobacter pylori eradication must be adapted to local resistance patterns, but the epidemiology of H. pylori resistance to antibiotics is poorly documented in Africa. The aim was to determine the antibiotic resistance rates, as well as the associated molecular mechanisms, of strains isolated in Dakar, Senegal.
Methods
One hundred and eight H. pylori strains were isolated between 2007 and 2009 from 108 patients presenting with upper abdominal pain to the Gastroenterology Department of Le Dantec Hospital. Antimicrobial susceptibility testing was performed for amoxicillin, clarithromycin, metronidazole, levofloxacin and tetracyclin using the E-test method. Mutations in the 23S rRNA gene of clarithromycin-resistant strains and in gyrA and gyrB of levofloxacin-resistant strains were investigated.
Results
Isolates were characterized by no resistance to amoxicillin (0%), tetracycline (0%), and very low rate of resistance to clarithromycin (1%), but a high rate of resistance to metronidazole (85%). The clarithromycin-resistant strain displayed the A2143G mutation. A worrying rate of levofloxacin resistance was detected (15%). N87I and D91N were the most common mutations in the quinolone-resistance-determining region of gyrA.
Conclusions
The first-line empirical regimen for H. pylori eradication in Senegal should include clarithromycin. Increasing rates of fluoroquinolone resistance detected should discourage the use of levofloxacin-containing regimens without prior antimicrobial susceptibility testing.
doi:10.1186/1476-0711-12-3
PMCID: PMC3552979  PMID: 23298145
Helicobacter pylori; Levofloxacin; Clarithromycin; Antibiotic resistance; Senegal
3.  Distribution of Spontaneous gyrA Mutations in 97 Fluoroquinolone-Resistant Helicobacter pylori Isolates Collected in France 
We determined the prevalence of gyrA mutations conferring fluoroquinolone resistance in 97 Helicobacter pylori isolates collected in France from 2007 to 2010. Ninety-four harbored one or two mutations already found in the quinolone resistance determining region (QRDR) of gyrA (for T87I, n = 23; for N87K, n = 32; for D91N, n = 30; for D91G, n = 7; for D91Y, n = 6), 2 harbored a mutation never previously described (D91H and A88P), and one strain was resistant (ciprofloxacin MIC of 8 mg/liter) without a detected mutation conferring this resistance in gyrA or gyrB genes.
doi:10.1128/AAC.05243-11
PMCID: PMC3256052  PMID: 22064536
4.  Evaluation of Four Commercial Real-Time PCR Assays for Detection of Bordetella spp. in Nasopharyngeal Aspirates ▿  
Journal of Clinical Microbiology  2011;49(11):3943-3946.
We evaluated the performances of 4 commercial real-time PCR kits for Bordetella pertussis IS481 sequence detection in nasopharyngeal aspirates by comparison with an in-house real-time PCR assay. Among them, the Simplexa Bordetella pertussis/parapertussis assay (Focus Diagnostics), the SmartCycler Bordetella pertussis/parapertussis assay (Cepheid), and Bordetella R-gene (Argene) present sensitivities over 90%. One kit proved unsuitable for routine clinical use.
doi:10.1128/JCM.00335-11
PMCID: PMC3209097  PMID: 21918018
5.  The rtxA Toxin Gene of Kingella kingae: a Pertinent Target for Molecular Diagnosis of Osteoarticular Infections▿† 
Journal of Clinical Microbiology  2011;49(4):1245-1250.
Kingella kingae is an emerging osteoarticular pathogen in young children. Its isolation by traditional culture methods remains difficult, underscoring the need to implement other diagnostic methods for its detection and identification, such as nucleic acid amplification tests. Although the genome of this bacterium has not yet been sequenced, a toxin named RTX has been identified. The goal of this study was to develop sensitive, specific, and rapid molecular methods based on the rtxA toxin gene sequence to diagnose this infection. Two real-time PCR assays (SYBR green and TaqMan chemistries) targeting this gene are reported. Sensitivity and specificity were first evaluated successfully with 67 strains: 31 Kingella kingae isolates and 36 strains from other bacterial species. Then, 52 clinical specimens positive or negative by culture and/or PCR (16S rRNA and cpn60 genes) were tested with these assays. A nested PCR assay with subsequent sequencing was also developed to confirm the presence of Kingella kingae isolates in these clinical specimens. The results obtained demonstrate that these assays are accurate for the diagnosis of Kingella kingae infection.
doi:10.1128/JCM.01657-10
PMCID: PMC3122863  PMID: 21248099
6.  Catabacter hongkongensis Bacteremia with Fatal Septic Shock 
Emerging Infectious Diseases  2011;17(7):1330-1331.
doi:10.3201/eid1707.101773
PMCID: PMC3381405  PMID: 21762611
bacteria; septic shock; bacteremia; Catabacter hongkongensis; letter
7.  Primary resistance to clarithromycin, metronidazole and amoxicillin of Helicobacter pylori isolated from Tunisian patients with peptic ulcers and gastritis: a prospective multicentre study 
Background
The frequency of primary resistance to antibiotics in H. pylori isolates is increasing worldwide. In Tunisia, there are limited data regarding the pattern of H. pylori antibiotic primary resistance.
Aim
To evaluate the primary resistance of H. pylori to clarithromycin, metronidazole and amoxicillin and to detect the mutations involved in clarithromycin resistance.
Materials and methods
273 strains isolated from adults and children were enrolled. The primary resistance to clarithromycin, metronidazole and amoxicillin was evaluated by means of E-test minimal inhibitory concentration (MIC). The real-time PCR using Scorpion primers was performed in all cases to assess clarithromycin primary resistance and point mutations involved.
Results
No resistance to amoxicillin was detected. For adults, resistance to clarithromycin and metronidazole was found respectively in 14.6% and 56.8%, and respectively in 18.8% and 25% in children. Overall, the rates of global primary resistance to clarithromycin and metronidazole in Tunisia were respectively determined in 15.4% and 51.3%.
By the use of Scorpion PCR, the A2143G was the most frequent point mutation observed (88.1%), followed by the A2142G (11.9%); the A2142C was not found and 18 of 42 patients (42.8%) were infected by both the resistant and the susceptible genotype.
The association of clarithromycin resistance with gender was not statistically significant, but metronidazole resistant strains were isolated more frequently in females (67.8%) than in males (32.2%) and the difference was significant. As for gastroduodenal diseases, the difference between strains isolated from patients with peptic ulceration and those with non peptic ulceration was not statistically significant. When about the distribution of resistant strains to clarithromycin and metronidazole between the three Tunisian cities (Tunis, Menzel Bourguiba and Mahdia), the difference was not statistically significant.
Conclusion
Local data regarding the primary resistance of H. pylori to clarithromycin, metronidazole and amoxicillin and the main genetic mutation involved in clarithromycin resistance in vivo (A2143G) are necessary to prove a clear need for a periodic evaluation of antibiotic consumption and new therapeutic strategies in Tunisia in order to avoid the emergence of resistant strains.
doi:10.1186/1476-0711-9-22
PMCID: PMC2928169  PMID: 20707901
8.  From array-based hybridization of Helicobacter pylori isolates to the complete genome sequence of an isolate associated with MALT lymphoma 
BMC Genomics  2010;11:368.
Background
elicobacter pylori infection is associated with several gastro-duodenal inflammatory diseases of various levels of severity. To determine whether certain combinations of genetic markers can be used to predict the clinical source of the infection, we analyzed well documented and geographically homogenous clinical isolates using a comparative genomics approach.
Results
A set of 254 H. pylori genes was used to perform array-based comparative genomic hybridization among 120 French H. pylori strains associated with chronic gastritis (n = 33), duodenal ulcers (n = 27), intestinal metaplasia (n = 17) or gastric extra-nodal marginal zone B-cell MALT lymphoma (n = 43). Hierarchical cluster analyses of the DNA hybridization values allowed us to identify a homogeneous subpopulation of strains that clustered exclusively with cagPAI minus MALT lymphoma isolates. The genome sequence of B38, a representative of this MALT lymphoma strain-cluster, was completed, fully annotated, and compared with the six previously released H. pylori genomes (i.e. J99, 26695, HPAG1, P12, G27 and Shi470). B38 has the smallest H. pylori genome described thus far (1,576,758 base pairs containing 1,528 CDSs); it contains the vacAs2m2 allele and lacks the genes encoding the major virulence factors (absence of cagPAI, babB, babC, sabB, and homB). Comparative genomics led to the identification of very few sequences that are unique to the B38 strain (9 intact CDSs and 7 pseudogenes). Pair-wise genomic synteny comparisons between B38 and the 6 H. pylori sequenced genomes revealed an almost complete co-linearity, never seen before between the genomes of strain Shi470 (a Peruvian isolate) and B38.
Conclusion
These isolates are deprived of the main H. pylori virulence factors characterized previously, but are nonetheless associated with gastric neoplasia.
doi:10.1186/1471-2164-11-368
PMCID: PMC3091627  PMID: 20537153
9.  Proficiency Program for Real-Time PCR Diagnosis of Bordetella pertussis Infections in French Hospital Laboratories and at the French National Reference Center for Whooping Cough and other Bordetelloses▿ † 
Journal of Clinical Microbiology  2009;47(10):3197-3203.
With the support of a ministerial program for innovative and expensive technologies, dedicated to the economic evaluation of laboratory diagnosis of pertussis by real-time PCR, external quality assessment for real-time IS481 PCR was carried out. Coordinated by the National Centre of Reference of Pertussis and other Bordetelloses (NCR), this study aimed to harmonize and to assess the performances of eight participating microbiology hospital laboratories throughout the French territory. Between January 2006 and February 2007, 10 proficiency panels were sent by the NCR (ascending proficiency program), representing a total of 49 samples and including eight panels to analyze and evaluate the global sensitivity and specificity of real-time PCR, one to assess the limit of detection, and one to evaluate nucleic acid extraction methods. As part of the descending proficiency program, extracted DNA from clinical samples was sent by the eight participating laboratories in different panels and analyzed by the NCR. In the ascending proficiency analysis, the sensitivity and specificity of the real-time PCR methods were 92.2% and 94.3%, respectively. The limit of detection of the different methods ranged between 0.1 and 1 fg/μl (0.2 to 2 CFU/μl). The nucleic acid extraction methods showed similar performances. During the descending proficiency analysis, performed with 126 samples, the result of the NCR for 15 samples (11.9%) was discordant with the result obtained by the source laboratory. Despite several initial differences, harmonization was easy and performances were homogeneous. However, the risk of false-positive results remains quite high, and we strongly recommend establishment of uniform quality control procedures performed regularly.
doi:10.1128/JCM.00817-09
PMCID: PMC2756922  PMID: 19692562
10.  Prevalence of Helicobacter pylori vacA, cagA, iceA and oipA genotypes in Tunisian patients 
Background
Distinct virulence factors of H. pylori have been described: the vaculating cytotoxin (vacA), the cytotoxin associated gene (cagA), the induced by contact with epithelium factor Antigen (iceA gene) and the outer membrane protein oipA. In Tunisia, there are no data regarding the pattern of H. pylori genotypes; therefore, this prospective and multicentre study was the first to be done in Tunisia and aimed to investigate the prevalence of the vacA, cagA, iceA and oipA genotypes of H. pylori isolates from Tunisian patients with peptic ulceration, gastric cancer, MALT lymphoma and gastritis.
Methods
H. pylori was cultured from endoscopic biopsies obtained from 281 Tunisian patients. The vacA alleles, cagA, iceA and oipA genotypes were determined by PCR.
Results
The vacA s1m1, s1m2 and s2m2 were respectively found in 10.7%, 12.5% and 45.6% of strains. The s2m1 genotype was not detected in our study. The cagA was found in 61.6% of isolates. The iceA1 and the iceA2 genotypes were respectively isolated in 60.2% and in 16% of strains. The oipA genotype was detected in 90.8% of strains. Considering the vacA and iceA genotypes, the presence of multiple H. pylori strains in a single biopsy specimen was found respectively in 31.4% and 23.8%. The comparison between strains isolated from antrum and fundus showed that Tunisian patients were infected with two or more strains of different cagA, vacA, iceA and oipA genotypes and the discordance was respectively in 9.6%, 4.6%, 8.9% and 8.5% of strains.
Conclusion
Our results showed that in 46% (131 strains among 281), the H. pylori strains were highly virulent in relation of the three or four virulent factors they could carry. These finding were described before in the literature. Tunisian patients were colonized by one or multiple strains of H. pylori in the same time in relation of presence of vacA m1/m2 and iceA1/iceA2 in the same biopsy. The discordance between strains isolated from antrum and fundus was high, and it is in favour of multicolonization.
doi:10.1186/1476-0711-9-10
PMCID: PMC2855517  PMID: 20302630
11.  Quadruplex Real-Time PCR Assay Using Allele-Specific Scorpion Primers for Detection of Mutations Conferring Clarithromycin Resistance to Helicobacter pylori ▿ †  
Journal of Clinical Microbiology  2008;46(7):2320-2326.
We developed a single-vessel multiplex real-time PCR assay that detects Helicobacter pylori infection and identified the four existing alleles of the 23S rRNA genes of H. pylori—the wild-type sequence and the three mutations conferring clarithromycin resistance—using allele-specific Scorpion primers directly on biopsy specimens. The Scorpion primers combine a primer and a probe in a single molecule and are able to distinguish single-nucleotide polymorphism. Fluorescent signals, produced when the probes are annealed, are read in four channels by a SmartCycler thermocycler. The assay was first applied successfully on 4 reference and 61 clinical strains. MICs of clarithromycin were determined by the Etest method. A perfect concordance was obtained between Etest and Scorpion PCR. Mixed populations were better detected by Scorpion PCR. We examined 259 biopsies from 229 patients by culture, PCR-restriction fragment length polymorphism (RFLP), and Scorpion PCR. One biopsy, positive for culture, exhibited inhibitors for both PCR-RFLP and Scorpion PCR. Twelve biopsies were positive for PCR-RFLP and Scorpion PCR but negative for culture with concordant determination of mutations in the 23S rRNA genes by the two PCR assays. Three biopsies were positive for Scorpion PCR only. Compared to culture, the sensitivity of Scorpion PCR was 98.3% and the specificity was 92.5%. The Scorpion PCR assay provides a highly accurate, rapid, and precise method for the detection and determination of mutations conferring clarithromycin resistance to H. pylori.
doi:10.1128/JCM.02352-07
PMCID: PMC2446943  PMID: 18463216
12.  Helicobacter pylori infection is not associated with an increased hemorrhagic risk in patients in the intensive care unit 
Critical Care  2006;10(3):R77.
Introduction
The potential role of Helicobacter pylori in acute stress ulcer in patients in an intensive care unit (ICU) is controversial. The aim of this study was to determine the frequency of H. pylori infection in ICU patients by antigen detection on rectal swabs, and to analyze the potential relationship between the presence of H. pylori and the risk of digestive gastrointestinal bleeding.
Methods
In this prospective, multicenter, epidemiological study, the inclusion criteria were as follows: patients admitted to the 12 participating ICU for at least two days, who were free of hemorrhagic shock and did not receive more than four units of red blood cells during the day before or the first 48 hours after admission to the ICU. Rectal swabs were obtained within the first 24 hours of admission to the ICU and were tested for H. pylori antigens with the ImmunoCard STAT! HpSA kit. The following events were analyzed according to H. pylori status: gastrointestinal bleeding, unexplained decline in hematocrit, and the number of red cell transfusions.
Results
The study involved 1,776 patients. Forty-nine patients (2.8%) had clinical evidence of upper digestive bleeding. Esophagogastroduodenoscopy was performed in 7.6% of patients. Five hundred patients (28.2%) required blood transfusion. H. pylori antigen was detected in 6.3% of patients (95% confidence interval 5.2 to 7.5). H. pylori antigen positivity was associated with female sex (p < 0.05) and with a higher Simplified Acute Physiology Score II (SAPS II; p < 0.05). H. pylori antigen status was not associated with the use of fiber-optic gastroscopy, the need for red cell transfusions, or the number of red cell units infused.
Conclusion
This large study reported a small percentage of H. pylori infection detected with rectal swab sampling in ICU patients and showed that the patients infected with H. pylori had no additional risk of gastrointestinal bleeding. Thus H. pylori does not seem to have a major role in the pathogenesis of acute stress ulcer in ICU patients.
doi:10.1186/cc4920
PMCID: PMC1550927  PMID: 16704741
14.  Performance Criteria of DNA Fingerprinting Methods for Typing of Helicobacter pylori Isolates: Experimental Results and Meta-Analysis 
Journal of Clinical Microbiology  1999;37(12):4071-4080.
Typing systems are used to discriminate between isolates of Helicobacter pylori for epidemiological and clinical purposes. Discriminatory power and typeability are important performance criteria of typing systems. Discriminatory power refers to the ability to differentiate among unrelated isolates; it is quantitatively expressed by the discriminatory index (DI). Typeability refers to the ability of the method to provide an unambiguous result for each isolate analyzed; it is quantitatively expressed by the percentage of typeable isolates. We evaluated the discriminatory power and the typeability of the most currently used DNA fingerprinting methods for the typing of H. pylori isolates: ribotyping, PCR-based restriction fragment length polymorphism (PCR-RFLP) analysis, and random amplified polymorphism DNA (RAPD) analysis. Forty epidemiologically unrelated clinical isolates were selected to constitute a test population adapted to the evaluation of these performance criteria. A meta-analysis of typeability and discriminatory power was conducted retrospectively with raw data from published studies in which ribotyping, PCR-RFLP, RAPD, repetitive extragenic palindromic DNA sequence-based PCR (REP-PCR), or pulsed-field gel electrophoresis (PFGE) was used. Experimental results and the meta-analysis demonstrated the optimal typeability (100%) and the excellent discriminatory powers of PCR-based typing methods: RAPD analysis, DIs, 0.99 to 1; REP-PCR, DI, 0.99; and PCR-RFLP analysis, DIs, 0.70 to 0.97). Chromosome restriction-based typing methods (ribotyping and PFGE) are limited by a low typeability (12.5 to 75%) that strongly decreases their discriminatory powers: ribotyping, DI, 0.92; PFGE, DIs, 0.24 to 0.88. We do not recommend the use of ribotyping and PFGE for the typing of H. pylori isolates. We recommend the use of PCR-based methods.
PMCID: PMC85883  PMID: 10565934
15.  Mutation in the peb1A Locus of Campylobacter jejuni Reduces Interactions with Epithelial Cells and Intestinal Colonization of Mice 
Infection and Immunity  1998;66(3):938-943.
Campylobacter jejuni is one of the leading causes of bacterial diarrhea throughout the world. We previously found that PEB1 is a homolog of cluster 3 binding proteins of bacterial ABC transporters and that a C. jejuni adhesin, cell-binding factor 1 (CBF1), if not identical to, contains PEB1. A single protein migrating at approximately 27 to 28 kDa was recognized by anti-CBF1 and anti-PEB1. To determine the role that the operon encoding PEB1 plays in C. jejuni adherence, peb1A, the gene encoding PEB1, was disrupted in strain 81-176 by insertion of a kanamycin resistance gene through homologous recombination. Inactivation of this operon completely abolished expression of CBF1, as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblotting. In comparison to the wild-type strain, the mutant strain showed 50- to 100-fold less adherence to and 15-fold less invasion of epithelial cells in culture. Mouse challenge studies showed that the rate and duration of intestinal colonization by the mutant were significantly lower and shorter than with the wild-type strain. In summary, PEB1 is identical to a previously identified cell-binding factor, CBF1, in C. jejuni, and the peb1A locus plays an important role in epithelial cell interactions and in intestinal colonization in a mouse model.
PMCID: PMC107999  PMID: 9488379

Results 1-15 (15)