Molecular epidemiology, antimicrobial susceptibilities and resistance mechanisms of Streptococcus pyogenes isolates resistant to erythromycin and tetracycline in Spain (1994-2006).

BACKGROUND: Group A Streptococcus (GAS) causes human diseases ranging in severity from uncomplicated pharyngitis to life-threatening necrotizing fasciitis and shows high rates of macrolide resistance in several countries. Our goal is to identify antimicrobial resistance in Spanish GAS isolates collected between 1994 and 2006 and to determine the molecular epidemiology (emm/T typing and PFGE) and resistance mechanisms of those resistant to erythromycin and tetracycline. RESULTS: Two hundred ninety-five out of 898 isolates (32.8%) were erythromycin resistant, with the predominance of emm4T4, emm75T25, and emm28T28, accounting the 67.1% of the 21 emm/T types. Spread of emm4T4, emm75T25 and emm28T28 resistant clones caused high rates of macrolide resistance. The distribution of the phenotypes was M (76.9%), cMLSB (20.3%), iMLSB (2.7%) with the involvement of the erythromycin resistance genes mef(A) (89.5%), msr(D) (81.7%), erm(B) (37.3%) and erm(A) (35.9%).Sixty-one isolates were tetracycline resistant, with the main representation of the emm77T28 among 20 emm/T types. To note, the combination of tet(M) and tet(O) tetracycline resistance genes were similar to tet(M) alone reaching values close to 40%. Resistance to both antibiotics was detected in 19 isolates of 7 emm/T types, being emm11T11 and the cMLSB phenotype the most frequent ones. erm(B) and tet(M) were present in almost all the strains, while erm(A), mef(A), msr(D) and tet(O) appeared in less than half of them. CONCLUSIONS: Spanish GAS were highly resistant to macrolides meanwhile showed minor resistance rate to tetracycline. A remarkable correlation between antimicrobial resistance and emm/T type was noticed. Clonal spread of emm4T4, emm75T25 and emm28T28 was the main responsable for macrolide resistance where as that emm77T28 clones were it to tetraclycline resistance. A wide variety of macrolide resistance genes were responsible for three macrolide resistance phenotypes.

Nalidixic acid disk for laboratory detection of ciprofloxacin resistance in Neisseria meningitidis.

Recently the CLSI recommended a disk diffusion method and breakpoints for meningococci which include breakpoints derived for nalidixic acid which serve as surrogate markers for gyrase A mutations associated with diminished fluoroquinolone susceptibility. This study presents the application of this methodology to a panel of 57 meningococcal strains isolated in Spain that include all levels of susceptibility to ciprofloxacin. In conclusion, the most useful method to predict isolates with gyrA mutations that decrease the activity of fluoroquinolones is the use of 30-microg nalidixic acid disks.

B:2a:p1.5 meningococcal strains likely arisen from capsular switching event still spreading in Spain.

Eighteen clustered cases of meningococcal disease associated with B:2a:P1.5 strains doubled the annual incidence up to 4.3 x 10(5) in Navarra, Spain, in 2007. Eleven percent of cases were fatalities, and 74% of cases were individuals 10 to 24 years old. This is the third cluster associated with this strain in northern Spain since 2001.

Emergence of high level azithromycin-resistant Neisseria gonorrhoeae strain isolated in Argentina.

One Neisseria gonorrhoeae strains highly resistant to azithromycin AzHLR (MIC >2048 mg/L) was isolated in Argentina in 2001 and it has been characterized by N. gonorrhoeae multiantigen sequence typing (NG-MAST) as ST696, suggesting a different event to other isolates in Europe. Neither, mtrR mutations or presence of mef gene were detected.

Antibiotic resistant meningococci in Europe: any need to act?

Meningococcal disease is a serious and rapidly progressing illness. It is therefore very important to monitor changes in the level of antibiotic susceptibility among clinical isolates. Different aspects such as interpretation of laboratory results, determination of breakpoints predicting treatment failure as well as definition of susceptibility levels in clinical samples using molecular methods are critical points for the surveillance of antibiotic resistance in Neisseria meningitidis. Within the strategic framework outlined by the EU.MenNet project, several objectives were identified to analyze 'The spread of antibiotic resistant meningococci in Europe', including the extent of antimicrobial resistance, its association with particular meningococcal lineages and geographical areas, as well as molecular characterization of the mechanisms involved, particularly in penicillin resistance. A heterogeneous figure for the frequency of intermediate resistance to penicillin appears across Europe. This heterogeneity may reflect different clonal lineages and/or uneven access to antibiotics in each country. In addition, the use of different criteria for the methodology used for definition cannot be avoided. The description of five specific changes associated with intermediate resistance to penicillin also allows the design of PCR-based tools to objectify results and for application in clinical samples.

Fluoroquinolone resistance in Neisseria meningitidis in Spain.

OBJECTIVES: To assess the ciprofloxacin resistance of Neisseria meningitidis isolated from 1999 through 2006 in Spain, susceptibility testing was conducted on 5300 isolates. METHODS: Ten isolates showed MICs of ciprofloxacin ranging between 0.06 and 0.25 mg/L, and they were characterized by analysis for mutations within the quinolone resistance determining regions (QRDRs) in the gyrA, gyrB, parC and parE genes. Mutations in the mtrR gene were also analysed. RESULTS AND CONCLUSIONS: Single mutations in gyrA appeared to be the main mechanism involved, Thr-91-->Ile being the most frequent substitution seen. Two meningococci had four different gyrA substitutions. No mutations in the QRDRs of the parC and gyrB genes were detected, and three strains showed a His-495-->Asn substitution in the parE gene. In addition, two different alterations in the mtrR gene affecting the expression of the MtrCDE efflux system were identified which may also contribute to the reduced susceptibility to quinolones seen in three strains.

Genotypes of Listeria monocytogenes strains isolated from 2000 to 2002 in Poland.

Pulsed field gel electrophoresis (PFGE), multiplex PCR and multilocus sequence typing (MLST) methods were used for genotyping study of seventy-three L. monocytogenes isolates collected in Poland between 2000 and 2002 from human, food, environment and a diseased goat. The multiplex PCR, which is an alternative method to classical serotyping, divided the isolates into four PCR groups, IIa (42.5%), IIb (27.4%), IIc (4.1%) and IVb (26%). The isolates displayed 33 distinct PFGE profiles. Twenty eight strains were further characterised by MLST based on sequence analyses of seven housekeeping genes. The combined sequence analyses revealed a total of 10 different allelic profiles from which 3 were not described earlier. It is intended that results obtained in this study will be the first data of a national database of L. monocytogenes genotypes in Poland.

Multicenter validation of a multiplex PCR assay for differentiating the major Listeria monocytogenes serovars 1/2a, 1/2b, 1/2c, and 4b: toward an international standard.

The performance of a multiplex PCR assay that separates the four major serovars of the pathogenic Listeria monocytogenes into four distinct PCR groups was evaluated through a multicenter typing study. Identical panels of 90 Listeria isolates were distributed to five participating laboratories that were blind to the nature of the isolates. Isolates were characterized using the previously standardized protocol. Overall concordance was 96.6 to 100%, sufficient for the assay to be used as an alternative to serotyping and confidently applied in laboratories involved in L. monocytogenes typing.

Antigenic and/or phase variation of PorA protein in non-subtypable Neisseria meningitidis strains isolated in Spain.

The PorA protein is a potential candidate as a vaccine component against meningococcal disease. However, this protein experiences antigenic variation and is subject to phase variations to evade immune selective pressure. In this study, the mechanisms responsible for altered expression of the PorA protein were analysed in 50 non-subtypable strains isolated from patients with meningococcal disease in Spain. The porA gene was amplified from 47 of the 50 strains. The majority of isolates were not recognized by the subtyping panel, as a result of non-synonymous base changes in the variable regions of the porA gene. Two of these strains revealed a premature stop codon before the variable region VR1 of PorA due to a single base-pair substitution at position 109 of the porA coding region. Another two presented a homopolymeric tract of eight adenine residues in the coding region, producing a DNA strand-slippage mechanism and PorA phase variation.

Molecular characterization of invasive serogroup Y Neisseria meningitidis strains isolated in the Latin America region.

OBJECTIVES: To improve the understanding of serogroup Y invasive meningococcal disease (IMD) in Latin America, particularly IMD molecular epidemiology; 166 Y serogroup isolates received at the National Reference Laboratories of Argentina, Brazil, Chile, Colombia, and Costa Rica during 2000-2006 were characterized by their molecular markers. METHODS: This analysis included serological assays to determine serogroup/serotype/serosubtype, DNA sequencing and genotyping of the porB and/or porA genes, multilocus sequence typing (MLST) and fetA allele determination. RESULTS: Sixteen different antigenic combinations were observed. Sixty-two (37.3%) isolates were NT:P1.5 and 36 (21.7%) isolates were 14:NST. Thirty-two different STs appeared, but 3 STs (ST-1624, ST-23, and ST-5770) accounted for 69.9% (116) of the strains. Most of the IMD isolates belonged to the ST-23, ST-167 clonal complexes or the group composed by ST-5770 and related STs. CONCLUSIONS: Isolates obtained in Colombia and Costa Rica were similar to that of the United States, in that most sequence types belonged to the ST-23 clonal complex. IMD isolates found in Argentina appear to be the result of an independent event and did not spread from nearby countries, being the sequence type ST-1624 (ST-167 clonal complex) the most frequently found. We were unable to correlate an antigenic shift of outer membrane proteins with an increase of serogroup Y meningococcal cases in our collection of isolates.

[Evolution of Neisseria meningitidis sensitivity to various antimicrobial drugs over the course of chemoprophylaxis during an epidemic outbreak]. / Evolución de la sensibilidad de Neisseria meningitidis a diversos antimicrobianos en el curso de intervenciones con quimioprofilaxis durante un brote epidémico.

INTRODUCTION: The aim of this study was to assess the evolution of the population MICs for various antimicrobial drugs against Neisseria meningitidis isolates obtained from asymptomatic carriers during a chemoprophylaxis campaign carried out for an epidemic outbreak of meningococcal disease in Nerva, a small town in Huelva province (Spain). MATERIAL AND METHODS: A nasopharyngeal carrier study including 427 people was carried out to determine the incidence rate of the epidemic strain among the general population. On the basis of the results, chemoprophylaxis with rifampicin was administered to the population aged 15 to 29 years (age group showing the highest incidence of the epidemic strain among carriers) living in Nerva. Three months later a new carrier study was performed (507 people) to evaluate the effects of chemoprophylaxis. Given the evolution of the outbreak, seven months later a new intervention was required with ciprofloxacin chemoprophylaxis; a second carrier study (399 people) was performed two months later to evaluate its effect. RESULTS: The number of strains isolated during the three carrier studies was 59 (13.8%), 33 (6.5%), and 22 (5.5%), respectively. Analysis of the changes in the MIC50 and MIC90 for the various antibiotics from the first to the second carrier study (rifampicin chemoprophylaxis) showed statistical differences only in the distribution of rifampicin MICs. Similarly, when changes from the second to the third study were analyzed (ciprofloxacin chemoprophylaxis), significant variations were detected for the cefotaxime MICs. Nevertheless, although there were changes in the MICs, the percentages of susceptibility from the beginning to the end of the study did not vary. CONCLUSIONS: Massive chemoprophylaxis in the age group with the highest incidence of the epidemic strain among carriers did not clearly modify the antibiotic susceptibility of the isolates. However, a slight increase in the MIC50 and MIC90 was observed for rifampicin after the first chemoprophylactic intervention and for cefotaxime at the end of the study. Consecutive chemoprophylactic interventions with rifampicin and ciprofloxacin had an impact on the percentage of meningococcal carriers in the overall population, with a clearly decreasing trend.

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.

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.

Sequencing of Neisseria meningitidis penA gene: the key to success in defining penicillin G breakpoints.

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 micro g/ml showed mosaic alleles, so 0.094 micro g/ml is suggested as the penicillin G intermediate breakpoint when E-test is used.

Evolución de la sensibilidad de Neisseria meningitidis a diversos antimicrobianos en el curso de intervenciones con quimioprofilaxis durante un brote epidémico / Evolution of Neisseria meningitidis sensitivity to various antimicrobial drugs over the course of chemoprophylaxis during an epidemic outbreak

Introducción. El objetivo del estudio fue evaluar la evolución de las concentraciones inhibitorias mínimas (CIM) poblacionales frente a diversos antimicrobianos en aislamientos de Neisseria meningitidis obtenidos de portadores asintomáticos en el contexto de un brote epidémico de enfermedad meningocócica localizado en la provincia de Huelva en el que se realizó una intervención de quimioprofilaxis en población general. Material y métodos. El primer estudio de portadores se realizó en 427 individuos con objeto de conocer la presencia de la cepa causante del brote en población general. Como resultado se procedió a utilizar quimioproflaxis con rifampicina en la población entre 15 y 29 años (grupo etario con mayor presencia de la cepa epidémica), residentes en la población de Nerva. A los 3 meses se realizó un nuevo estudio de portadores (507 personas) para evaluar el efecto de dicha quimioprofilaxis. Dada la evolución del brote epidémico fue necesario realizar a los 7 meses una nueva intervención con ciprofloxacino y transcurridos 2 meses un nuevo estudio de portadores (399 personas) para evaluar su efecto. Resultados. El número de cepas aisladas en los 3 estudios de portadores realizados fue de 59 (13,8%), 33 (6,5%) y 22 (5,5%), respectivamente. El análisis de la evolución de la CIM50 y CIM90 de los distintos antibióticos del primer al segundo estudio de portadores (quimioprofilaxis con rifampicina) únicamente detectó cambios con significación estadística en las CIM de rifampicina. Así mismo, cuando se analizó la variación en la distribución de las CIM del segundo al tercer estudio (quimioprofilaxis con ciprofloxacino) fueron detectados cambios significativos particularmente en las CIM de cefotaxima. Aunque existieron variaciones de CIM, los porcentajes de sensibilidad al principio y final del estudio no variaron. Conclusiones. El empleo de quimioprofilaxis masiva en el grupo de edad con mayor porcentaje de portadores de la cepa de N. meningitidis responsable del brote no modificó sensiblemente la sensibilidad antibiótica de los aislados, si bien puede observarse un aumento de los valores de la CIM50 y CIM90 en el caso de la rifampicina tras la intervención con este antimicrobiano, y en el caso de la cefotaxima tras las 2 intervenciones realizadas. Las sucesivas intervenciones de quimioprofilaxis con rifampicina y posteriormente con ciprofloxacino tuvieron un claro reflejo en el porcentaje portadores en la población general, con una tendencia claramente decreciente (AU)

Introduction. The aim of this study was to assess the evolution of the population MICs for various antimicrobial drugs against Neisseria meningitidis isolates obtained from asymptomatic carriers during a chemoprophylaxis campaign carried out for an epidemic outbreak of meningococcal disease in Nerva, a small town in Huelva province (Spain). Material and methods. A nasopharyngeal carrier study including 427 people was carried out to determine the incidence rate of the epidemic strain among the general population. On the basis of the results, chemoprophylaxis with rifampicin was administered to the population aged 15 to 29 years (age group showing the highest incidence of the epidemic strain among carriers) living in Nerva. Three months later a new carrier study was performed (507 people) to evaluate the effects of chemoprophylaxis. Given the evolution of the outbreak, seven months later a new intervention was required with ciprofloxacin chemoprophylaxis; a second carrier study (399 people) was performed two months later to evaluate its effect. Results. The number of strains isolated during the three carrier studies was 59 (13.8%), 33 (6.5%), and 22 (5.5%), respectively. Analysis of the changes in the MIC50 and MIC90 for the various antibiotics from the first to the second carrier study (rifampicin chemoprophylaxis) showed statistical differences only in the distribution of rifampicin MICs. Similarly, when changes from the second to the third study were analyzed (ciprofloxacin chemoprophylaxis), significant variations were detected for the cefotaxime MICs. Nevertheless, although there were changes in the MICs, the percentages of susceptibility from the beginning to the end of the study did not vary. Conclusions. Massive chemoprophylaxis in the age group with the highest incidence of the epidemic strain among carriers did not clearly modify the antibiotic susceptibility of the isolates. However, a slight increase in the MIC50 and MIC90 was observed for rifampicin after the first chemoprophylactic intervention and for cefotaxime at the end of the study. Consecutive chemoprophylactic interventions with rifampicin and ciprofloxacin had an impact on the percentage of meningococcal carriers in the overall population, with a clearly decreasing trend (AU)