Molecular characterization of ciprofloxacin resistance of gonococcal strains in Spain.

BACKGROUND: Over the past several years, the emergence of gonococcal isolates with intermediate or full resistance to fluoroquinolones has become a significant concern in several countries, including Spain. GOAL: The goal was to determine the occurrence of ciprofloxacin resistance among Neisseria gonorrhoeae strains in Spain during 2000 to 2001 and determine the frequency and patterns of mutations at gyrA, gyrB, and parC genes in these isolates. STUDY DESIGN: Eleven ciprofloxacin-resistant strains (with MICs ranging from 1 to 64 micrograms/mL) and two intermediate isolates (with MICs of 0.12 and 0.5 microgram/mL) were found. Mutations were identified by polymerase chain reaction and direct sequencing of the amplified products. RESULTS AND CONCLUSIONS: Alterations at Ser-91 and Asp-95 in GyrA were detected in all strains except one, an isolate for which the MIC was 0.12 microgram/mL. Alterations in ParC were more variable, and there was no clear correlation between the number of parC mutations and the level of resistance. No alterations at gyrB gene associated with ciprofloxacin resistance were found. The resistance was distributed among different types of strains, suggesting that the increase in the incidence of ciprofloxacin-resistant strains in Spain was not exclusively due to the appearance of a single-strain outbreak.

Development of a multilocus sequence typing method for analysis of Listeria monocytogenes clones.

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.

Impact of meningococcal vaccination with combined serogroups A and C polysaccharide vaccine on carriage of Neisseria meningitidis C.

Two studies of meningococcal carriage state were carried out in Galicia (Spain) before and after a mass vaccination campaign between December 1996 and January 1997 against Neisseria meningitidis serogroup C with meningococcal serogroups A and C polysaccharide vaccine. The studies covered two areas with different incidence rates of meningococcal disease in 1996 (high and low incidence). Carriage rates of serogroup C showed a decrease in both areas, 47 and 65 % respectively, before and after the vaccination. Results showed a decrease in carrier state in the age groups 10-14- and 15-19-year-olds, but not in the 5-9-year-olds. These results demonstrate the effect of immunization on the reduction of the carriage state.

Antibiotic resistance of Neisseria gonorrhoeae in Spain: trends over the last two decades.

In vitro activities of six antimicrobial agents against 2966 strains of Neisseria gonorrhoeae, isolated in Spain between 1983 and 2001, were determined. The percentages of intermediately susceptible and resistant isolates to penicillin (MIC > or = 0.12 mg/L) and tetracycline (MIC > or = 0.5 mg/L) were very high over the period of study. Strains intermediately susceptible to cefoxitin were identified at a variable percentage during the study. All N. gonorrhoeae isolates were susceptible to spectinomycin and ceftriaxone. Recently, resistance to ciprofloxacin has emerged.

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.

What about antibiotic resistance in Neisseria lactamica?

The in vitro activity of penicillin, ampicillin, cefotaxime, ceftriaxone, rifampicin and ciprofloxacin against 286 Neisseria lactamica isolates was determined by agar dilution and the category of susceptibility was analysed in accordance with the criteria used for Neisseria meningitidis. All isolates were considered to have intermediate susceptibility to penicillin. A total of 1.7% of the isolates were resistant to ampicillin but all were susceptible to cefotaxime and ceftriaxone. Rifampicin MICs ranged between 0.12 and 2 mg/L. Six isolates (2.1%) showed decreased susceptibility to ciprofloxacin.

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).

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.

[Susceptibility to antimicrobial drugs used in the prophylaxis of meningococcal disease: situation after an epidemic wave]. / Sensibilidad a los principales antimicrobianos empleados en las profilaxis antimeningocócica: situación tras una onda epidémica.

In the early 1990s a rise in the incidence of meningococcal disease was observed in Galicia, Spain, most cases of which were caused by serogroup C meningococcal strains. As part of the epidemiological analysis of this epidemic wave, two studies of asymptomatic carriers of neisseria meningitidis were carried out: the first took place during the period of maximum incidence and coincided with a massive immunization campaign (December 1996 to January 1997); and the second was conducted one year later (January 1998). A total of 1234 meningococcal strains were isolated in both studies (789 in the first and 445 in the second study) and the susceptibility to rifampin, ciprofloxacin, ceftriaxone and sulfadiazine was determined. The susceptibility to rifampin, ciprofloxacin and cetriaxone was high among the strains isolated in both studies. For sulfadiazine, the percentage of resistant strains was 92.6% for the first and 86.3% for the second study.

Sensibilidad a los principales antimicrobianos empleados en la profilaxis antimeningocócica: situación tras una onda epidémica / Susceptibility to antimicrobial drugs used in the prophylaxis of meningococcal disease: Situation after an epidemic wave

En la segunda mitad de la década de 1990 se produjo en Galicia un importante aumento en la incidencia de enfermedad meningocócica, siendo la mayoría de los casos producidos por cepas del serogrupo C. Como parte de la investigación epidemiológica de esta onda epidémica se realizaron dos amplios estudios de portadores asintomáticos de neisseria meningitidis. El primero tuvo lugar durante el periodo de máxima incidencia de la enfermedad, coincidiendo con una campaña masiva de vacunación (diciembre 1996-enero 1997); el segundo se realizó un año más tarde (enero 1998). En las 1234 cepas de meningococo aisladas de ambos estudios (789 del estudio 96/97 y 445 del estudio 98) se determinó la sensibilidad a los antibióticos utilizados en la profilaxis antimeningocócica: rifampicina, ciprofloxacino, ceftriaxona y sulfadiazina. La sensibilidad a rifampicina, ciprofloxacino y ceftriaxona fue elevada en las cepas procedentes de ambos estudios. En cuanto a la sulfadiazina, el porcentaje de cepas resistentes se situó en un 92,6 por ciento en el estudio 96/97 y en un 86,3 por ciento en el estudio 98 (AU)

Antibiotic susceptibility patterns of Neisseria meningitidis isolates from patients and asymptomatic carriers.

The activities of seven antimicrobial agents used for treatment and prophylaxis of meningococcal disease was investigated against 901 Neisseria 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.

Carriage of a new epidemic strain of Neisseria meningitidis and its relationship with the incidence of meningococcal disease in Galicia, Spain.

In Galicia, Spain, a dramatic increase in the incidence of meningococcal disease was seen in the 1995-6. The annual incidence rose to 11 per 10(5) inhabitants, and 80% of identified strains were C:2b:P1.2,5. This led to the implementation of an intensive A+C vaccination campaign for the population aged 18 months to 19 years. During this campaign the prevalence of carriage in areas with high and low incidence was studied. Nasopharyngeal swabs were taken from 9796 subjects immediately before the administration of meningococcal vaccine, plated onto Thayer-Martin plates, incubated and sent for analysis to the Reference Laboratory for Neisseria in Spain. The prevalence of the C:2b: P1.2,5 strains was 0.6% (95% CI 0.29-0.88) in the high incidence area, and 0.41% (95 % CI 0.00-1.04) in the low incidence area, and that of serogroup C (all strains) 1.36% (95% CI 0.80-1.80) and 0.89% (95% CI 0.09-1.69) respectively. The prevalence of N. meningitidis (all strains) was almost the same in both areas (8%). Carriers of the epidemic strain were not found in the 2-4 year age group, that most affected by the disease. Our data showed a wide distribution but a low carriage rate of the epidemic strain C:2b:P1.2,5 in the high and low disease incidence areas studied; the difference in the carriage rates between the two areas was not statistically significant.