Luisa Arreaza

Development of a Multilocus Sequence Typing Method for Analysis of Listeria monocytogenes Clones

ABSTRACT This study is a first step in the development of multilocus sequence typing (MLST) method for Listeria monocytogenes. Nine housekeeping genes were analyzed in a set of 62 strains isolated from different sources and geographic locations in Spain. These strains were previously characterized by pulsed-field gel electrophoresis (PFGE). Because of low diversity, two loci were discarded from the study. The sequence analysis of the seven remaining genes showed 29 different allelic combinations, with 22 of them represented by only one strain. The results of this sequence analysis were generally consistent with those of PFGE. Because MLST allows the easy comparison and exchange of results obtained in different laboratories, the future application of this new molecular method could be a useful tool for the listeriosis surveillance systems that will allow the identification and distribution of analysis of L. monocytogenes clones in the environment.

Interlaboratory comparison of agar dilution and etest methods for determining the MICs of antibiotics used in management of Neisseria meningitidis infections

ABSTRACT Previous studies have shown that there is considerable variation in the methods and media used to determine the susceptibility of Neisseria meningitidis to antimicrobial agents in different countries. In this study, national and regional reference laboratories used a standardized methodology to determine the MICs of antibiotics used in the management of meningococcal infection. Fourteen laboratories participated in the study, determining the susceptibility to penicillin G, rifampin, cefotaxime, ceftriaxone, ciprofloxacin, and ofloxacin of a collection of 17 meningococci, of which 11 strains were previously defined as having intermediate resistance to penicillin (PenI) by sequencing and restriction fragment length polymorphism analysis of the penA gene. The MIC was determined by agar dilution and Etest with Mueller-Hinton agar (MH), MH supplemented with sheep blood (MH+B), and MH supplemented with heated (chocolated) blood. Several laboratories encountered problems obtaining confluent growth with unsupplemented MH. MH+B was considered to give the most congruent and reproducible results among the study laboratories. The modal MIC for MH+B for each antibiotic and method was calculated to define the MIC consensus, allowing assessment of each individual laboratorys data in relation to the others. The agreement in each antibiotic/method/medium combination was defined as the percentage of laboratories with a result within one dilution of the modal result. For the whole study, an agreement of 90.6% was observed between agar dilution and Etest methods. The agreement in each laboratory/antibiotic/method combination ranged from 98.2% to 69.7%, with six laboratories demonstrating agreement higher than 90% and 11 more than 80%. The ability of the laboratories to detect the PenI isolates ranged from 18.2% to 100%. The apparent difficulty in interpreting susceptibility to rifampin, particularly with the Etest method, is very interesting.

Capsule Switching among C:2b:P1.2,5 Meningococcal Epidemic Strains after Mass Immunization Campaign, Spain

A mass immunization campaign for 18-month to 19-year-olds was undertaken in Spain in 1996–1997 because of an epidemic of serogroup C meningococcal disease associated with a C:2b:P1.2,5 strain belonging to the A4 lineage. Surveillance for the “capsule-switching” phenomenon producing B:2b:P1.2,5 isolates was undertaken. Of 2,975 meningococci characterized, B:2b:P1.2,5 and B:2b:P1.2 antigenic combinations were found in 18 isolates; 15 meningococci were defined as serogroup B belonging to the A4 lineage.

Antibiotic Susceptibility Patterns of Neisseria meningitidis Isolates from Patients and Asymptomatic Carriers

ABSTRACT The activities of seven antimicrobial agents used for treatment and prophylaxis of meningococcal disease was investigated against 901Neisseria meningitidis isolates, 112 of which were recovered from patients and 789 of which were recovered from asymptomatic carriers. The proportions of isolates with decreased susceptibility to penicillin were 55.3 and 39.0%, respectively. Penicillin- and ampicillin-intermediate strains were more common among serogroup C meningococci than among non-serogroup C meningococci from both patients and carriers.

The epidemic wave of meningococcal disease in Spain in 1996-1997: probably a consequence of strain displacement

During 1996 and 1997 an epidemic wave of meningococcal disease took place in Spain. Initial studies described the antigenic expression of the epidemic strain as C:2b:P1.2,5 and proposed that it was a variant of the previously identified Spanish C:2b:non-subtypable epidemic strain. To clarify this hypothesis, 1036 C:2b:P1.2(5) and 76 C:2b:NST isolates obtained during 1992-1999 were analysed by pulsed-field gel electrophoresis. The majority of the C:2b:P1.2,5 and C:2b:P1.2 isolates showed one of two very closely related profiles. During the epidemic period, 80% of the C:2b:NST strains showed these two pulsotypes. However, before the epidemic wave, most of these C:2b:NST strains (60%) showed a profile that was found infrequently among C:2b:P1.2,5 and C:2b:P1.2 isolates. A similar evolution was observed in C:2b:P1.5 isolates. Thirty-four C:2b:P1.2(5) and 10 C:2b:NST isolates, exhibiting representative pulsotypes, were subjected to multi-locus sequence typing. Isolates belonging to both A4 and ET-37 lineages were identified. These data point to the possibility that the A4 cluster has displaced the ET-37 complex among serogroup C meningococci in Spain.

Sequencing of Neisseria meningitidis penA Gene: the Key to Success in Defining Penicillin G Breakpoints

ABSTRACT Testing of susceptibility to penicillin G by E-test and sequencing of an internal fragment of the penA gene were done for 43 meningococcal strains. Those strains for which the MIC was ≥0.094 μg/ml showed mosaic alleles, so 0.094 μg/ml is suggested as the penicillin G intermediate breakpoint when E-test is used.

Dynamics of the penA Gene in Serogroup C Meningococcal Strains

The transpeptidase encoding region of the penA gene was sequenced in 44 meningococcal strains (41 serogroup C [23 characterized as serotype 2b and 18 as serotype 2a] and 3 serogroup B [B:2b:P1.2,5]). All strains were characterized by multilocus sequence typing and were determined to be susceptible or intermediate resistant to penicillin (Pen(s) or Pen(i), respectively). A high degree of homology was found among the penA alleles identified in the Pen(s) strains. All the Pen(i) C:2b strains, which belonged to 2 different clonal complexes, showed the same penA gene allele. This fact suggests that 1 of the clonal complexes acquired that allele, spreading it to the other by horizontal transfer. The same allele also was found in the B:2b strains studied, indicating that 1 of the Pen(i) C:2b strains underwent a capsular switching event. A different mosaic penA allele was identified in the Pen(i) C:2a strains, which belonged to the ET37 cluster.

Investigation for a more virulent variant among the C:2b:P1.2,5 Spanish meningococcal epidemic strains by molecular epidemiology.

A rise in the incidence of meningococcal disease has occurred in Spain in recent years, especially in some regions in the north-west of the country. Most cases have been caused by meningococci characterised as Neisseria meningitidis C:2b:P1.2,5. A total of 107 C:2b:P1.2,5 meningococcal isolates (60 from patients and 47 from carriers) and 12 isolates showing related antigenic combinations (C:2b:NST, C:2b:P1.2, C:2b:P1.5, C:NT:P1.2,5) was analysed by pulsed-field gel electrophoresis to determine the genetic variability of the epidemic and related strains. Endonucleases BglII and NheI were used to cut chromosomal DNA. When BglII was used, most of the C:2b:P1.2,5 isolates showed the same pulsotype regardless of whether they were from clinical cases or carriers. Isolates showing the principal profile after digestion with endonuclease BglII were analysed with NheI. Four pulsotypes were identified, of which two were found in only one isolate each. The major profiles (1 and 2) showed differential distribution among clinical and carrier isolates; pulsotype 1 was the most frequent among clinical isolates. However, the proportions of isolates showing profiles 1 and 2 were similar among carrier isolates. This could indicate that there are two variants of the C:2b:P1.2,5 strain with differing pathogenicity.

Molecular Approach for the Study of Penicillin Resistance In Neisseria meningitidis

Neisseria meningitidis was previously considered extremely susceptible to penicillin, with most isolates showing minimal inhibitory concentrations (MICs) of ≦ 0.06 μg/mL. However, meningococcal isolates with decreased susceptibility to penicillin have been reported from asymptomatic carriers from as long ago as 1964 (1). Since then, meningococcal clinical isolates with decreased susceptibility to penicillin have been widely described in different countries, with MICs between 0.12 μg/mL to 1 μg/mL (2-9).