Eric Hernandez

Variable Number of Tandem Repeats in Salmonella enterica subsp. enterica for Typing Purposes

ABSTRACT The genomic sequences of Salmonella enterica subsp. enterica strains CT18, Ty2 (serovar Typhi), and LT2 (serovar Typhimurium) were analyzed for potential variable number tandem repeats (VNTRs). A multiple-locus VNTR analysis (MLVA) of 99 strains of S. enterica supsp. enterica based on 10 VNTRs distinguished 52 genotypes and placed them into four groups. All strains tested were independent human isolates from France and did not reflect isolates from outbreak episodes. Of these 10 VNTRs, 7 showed variability within serovar Typhi, whereas 1 showed variability within serovar Typhimurium. Four VNTRs showed high Neis diversity indices (DIs) of 0.81 to 0.87 within serovar Typhi (n = 27). Additionally, three of these more variable VNTRs showed DIs of 0.18 to 0.58 within serovar Paratyphi A (n = 10). The VNTR polymorphic site within multidrug-resistant (MDR) serovar Typhimurium isolates (n = 39; resistance to ampicillin, chloramphenicol, spectinomycin, sulfonamides, and tetracycline) showed a DI of 0.81. Cluster analysis not only identified three genetically distinct groups consistent with the present serovar classification of salmonellae (serovars Typhi, Paratyphi A, and Typhimurium) but also discriminated 25 subtypes (93%) within serovar Typhi isolates. The analysis discriminated only eight subtypes within serovar Typhimurium isolates resistant to ampicillin, chloramphenicol, spectinomycin, sulfonamides, and tetracycline, possibly reflecting the emergence in the mid-1990s of the DT104 phage type, which often displays such an MDR spectrum. Coupled with the ongoing improvements in automated procedures offered by capillary electrophoresis, use of these markers is proposed in further investigations of the potential of MLVA in outbreaks of salmonellosis, especially outbreaks of typhoid fever.

Antibiotic Susceptibilities of 96 Isolates of Bacillus anthracis Isolated in France between 1994 and 2000

ABSTRACT Ninety-six isolates of Bacillus anthracis recovered in France between 1994 and 2000 were tested for their susceptibilities to 25 different antibiotics. Resistance to penicillin G and amoxicillin was 11.5%. All of the isolates were resistant to cotrimoxazole and susceptible to doxycycline, ciprofloxacin, pefloxacin, levofloxacin, teicoplanin, vancomycin, clindamycin, imipenem, and rifampin.

Ineffectiveness of the Binax NOW malaria test for diagnosis of Plasmodium ovale malaria.

Malaria remains the most dangerous tropical disease for the French troops deployed overseas. In 2003, 768 cases in the French army were declared along with an increase of incidence of 400% in comparison with 2002, mainly due to the Licorne peacekeeping operation in Ivory Coast. In order to ensure fast diagnosis in the field, a rapid immunochromatographic test (NOW malaria test; Binax, Portland, Oreg.) has been provided to military doctors. This test detects the Plasmodium falciparum-specific HRP2 antigen and a panmalarial aldolase common to all species. The assay is sensitive for the detection of P. falciparum (1) and Plasmodium vivax (2) but, in our experience, is poorly sensitive for the detection of Plasmodium ovale, a species involved in cases of malaria among the French troops in western Africa. Between November 2002 and August 2004, 114 samples from patients presenting a malaria attack were analyzed in the laboratory of the French military hospital of Begin (Saint-Mande, France). The Plasmodium species identified were P. falciparum (n = 93), P. ovale (n = 12), P. vivax (n = 9), and P. malariae (n = 1). For each specimen, thin and thick blood films, the Binax NOW malaria test, and a specific SYBR green real-time PCR using the Lightcycler instrument (Roche Diagnostics, Meylan, France) were used for diagnosis. This technique is considered the “gold standard” in our laboratory. Sequences used for the design of primers were the 18S RNA genes of the four species (Table ​(Table1),1), which have been previously validated (data not shown). Thin and thick blood films were stained with a rapid coloration set (Diff-Quick; Dade Behring, Newark, Del.) and examined by two experienced microscopists during 20 min. The rapid immunochromatographic test was used according to the manufacturers recommendations. TABLE 1. Comparison of Binax NOW malaria test versus microscopic examination and PCR Among the 22 patients infected with non-P. falciparum species, 9 were positive for P. vivax, 12 were positive for P. ovale, and 1 was positive for P. malariae by microscopic examination (Table ​(Table1).1). PCR was used to detect all the infections and confirm all microscopic identifications. The Binax NOW malaria test detected all cases of P. vivax infection (9 of 9) but only 3 of the 12 cases of P. ovale infection (25%). The test was positive in the only case of P. malariae infection. With P. falciparum, 89 cases were positive, and two false positives and one false negative were found. Considering these data, it seems that the Binax NOW malaria test is not reliable for the detection of P. ovale infection. This has been previously described in a study including nine cases of P. ovale infection (2). The inability of the rapid immunochromatographic test to detect P. ovale has been also observed with the ICT Malaria P.f/P.v test (3, 4). The main explanation for the failure of the assay was low parasite density, but in this study all infections due to P. ovale were detected by microscopic examination. The inaccuracy could be due to low production of the aldolase by P. ovale or, as supposed by Mason et al., to regional variations in the genetic determinants of ICT panmalarial antigen (5). In case of suspicion of malaria challenge, the diagnosis of infection with P. ovale must not be elicited, and blood smear examination remains necessary for the elimination of the diagnosis.

Susceptibility of 71 French isolates of Francisella tularensis subsp. holarctica to eight antibiotics and accuracy of the Etest® method

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Comparison of Minisatellite Polymorphisms in the Bacillus cereus Complex: a Simple Assay for Large-Scale Screening and Identification of Strains Most Closely Related to Bacillus anthracis

ABSTRACT Polymorphism of five tandem repeats that are monomorphic in Bacillus anthracis was investigated in 230 isolates of the B. cereus group and in 5 sequenced B. cereus genomes in search for markers allowing identification of B. cereus and B. thuringiensis strains most closely related to B. anthracis. Using this multiple-locus variable number of tandem repeat analysis (MLVA), a cluster of 30 strains was selected for further characterization. Eventually, six of these were characterized by multilocus sequence type analysis. One of the strains is only six point mutations (of almost 3,000 bp) away from B. anthracis and was also proposed to be closest to B. anthracis by MLVA analysis. However, this strain remains separated from B. anthracis by a number of significant genetic events observed in B. anthracis, including the loss of the hemolysin activity, the presence of four prophages, and the presence of the two virulence plasmids, pXO1 and pXO2. One particular minisatellite marker provides an efficient assay to identify the subset of B. cereus and B. thuringiensis strains closely related to B. anthracis. Based on these results, a very simple assay is proposed that allows the screening of hundreds of strains from the B. cereus complex, with modest equipment and at a low cost, to eventually fill the gap with B. anthracis and better understand the origin and making of this dangerous pathogen.