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Antibiotic susceptibility and resistance in Neisseria meningitidis: phenotypic and genotypic characteristics
Örebro University, School of Health and Medical Sciences.
2009 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Neisseria meningitidis, also known as the meningococcus, is a globally spread obligate human bacterium causing meningitis and/or septicaemia. It is responsible for epidemics in both developed and developing countries. Untreated invasive meningococcal disease is often fatal, and despite modern intensive care units, the mortality is still remarkably high (approximately 10%). The continuously increasing antibiotic resistance in many bacterial pathogens is a serious public health threat worldwide and there have been numerous reports of emerging resistance in meningococci during the past decades.

In paper I, the gene linked to reduced susceptibility to penicillins, the penA gene, was examined. The totally reported variation in all published penA genes was described. The penA gene was highly variable (in total 130 variants were identified). By examination of clinical meningococcal isolates, the association between penA gene sequences and penicillin susceptibility could be determined. Isolates with reduced susceptibility displayed mosaic structures in the penA gene. Two closely positioned nucleotide polymorphisms were identified in all isolates with reduced penicillin susceptibility and mosaic structured penA genes. These alterations were absent in all susceptible isolates and were successfully used to detect reduced penicillin susceptibility by real-time PCR and pyrosequencing in paper II. In papers III and IV, antibiotic susceptibility and characteristics of Swedish and African meningitis belt meningococcal isolates were comprehensively described. Although both populations were mainly susceptible to the antibiotics used for treatment and prophylaxis, the proportion of meningococci with reduced penicillin susceptibility was slightly higher in Sweden. A large proportion of the African isolates was resistant to tetracycline and erythromycin. In paper V, the gene linked to rifampicin resistance, the rpoB gene, was examined in meningococci from 12 mainly European countries. Alterations of three amino acids in the RpoB protein were found to always and directly lead to rifampicin resistance. A new breakpoint for rifampicin resistance in meningococci was suggested. The biological cost of the RpoB alterations was investigated in mice. The pathogenicity/virulence was significantly lower in rifampicin resistant mutants as compared with susceptible wild-type bacteria.

Place, publisher, year, edition, pages
Örebro: Örebro universitet , 2009. , p. 94
Series
Örebro Studies in Medicine, ISSN 1652-4063 ; 38
Keywords [en]
Neisseria meningitidis, meningococcal disease, antibiotic resistance, antibiotic susceptbility, biological cost, PCR, sequencing
National Category
Cell and Molecular Biology Microbiology in the medical area Microbiology in the medical area Microbiology in the medical area
Research subject
Biomedicine; Medicine
Identifiers
URN: urn:nbn:se:oru:diva-8652ISBN: 978-91-7668-702-4 (print)OAI: oai:DiVA.org:oru-8652DiVA, id: diva2:278317
Public defence
2009-12-18, Wilandersalen, Universitetssjukhuset Örebro, Örebro, 09:00 (English)
Opponent
Supervisors
Available from: 2009-11-25 Created: 2009-11-25 Last updated: 2018-01-12Bibliographically approved
List of papers
1. Total variation in the penA gene of Neisseria meningitidis: correlation between susceptibility to beta-lactam antibiotics and penA gene heterogeneity
Open this publication in new window or tab >>Total variation in the penA gene of Neisseria meningitidis: correlation between susceptibility to beta-lactam antibiotics and penA gene heterogeneity
2006 (English)In: Antimicrobial Agents and Chemotherapy, ISSN 0066-4804, E-ISSN 1098-6596, Vol. 50, no 10, p. 3317-3324Article in journal (Refereed) Published
Abstract [en]

In recent decades, the prevalence of Neisseria meningitidis isolates with reduced susceptibility to penicillins has increased. The intermediate resistance to penicillin (Pen(i)) for most strains is due mainly to mosaic structures in the penA gene, encoding penicillin-binding protein 2. In this study, susceptibility to beta-lactam antibiotics was determined for 60 Swedish clinical N. meningitidis isolates and 19 reference strains. The penA gene was sequenced and compared to 237 penA sequences from GenBank in order to explore the total identified variation of penA. The divergent mosaic alleles differed by 3% to 24% compared to those of the designated wild-type penA gene. By studying the final 1,143 to 1,149 bp of penA in a sequence alignment, 130 sequence variants were identified. In a 402-bp alignment of the most variable regions, 84 variants were recognized. Good correlation between elevated MICs and the presence of penA mosaic structures was found especially for penicillin G and ampicillin. The Pen(i) isolates comprised an MIC of >0.094 microg/ml for penicillin G and an MIC of >0.064 microg/ml for ampicillin. Ampicillin was the best antibiotic for precise categorization as Pen(s) or Pen(i). In comparison with the wild-type penA sequence, two specific Pen(i) sites were altered in all except two mosaic penA sequences, which were published in GenBank and no MICs of the corresponding isolates were described. In conclusion, monitoring the relationship between penA sequences and MICs to penicillins is crucial for developing fast and objective methods for susceptibility determination. By studying the penA gene, genotypical determination of susceptibility in culture-negative cases can also be accomplished.

National Category
Medical and Health Sciences
Research subject
Medicine
Identifiers
urn:nbn:se:oru:diva-8529 (URN)10.1128/AAC.00353-06 (DOI)17005811 (PubMedID)
Available from: 2009-11-25 Created: 2009-11-12 Last updated: 2017-12-12Bibliographically approved
2. Combined real-time PCR and pyrosequencing strategy for objective, sensitive, specific, and high throughput identification of reduced susceptibility to penicillins in Neisseria meningitidis
Open this publication in new window or tab >>Combined real-time PCR and pyrosequencing strategy for objective, sensitive, specific, and high throughput identification of reduced susceptibility to penicillins in Neisseria meningitidis
2008 (English)In: Antimicrobial Agents and Chemotherapy, ISSN 0066-4804, E-ISSN 1098-6596, Vol. 52, no 2, p. 753-756Article in journal (Refereed) Published
Abstract [en]

A segment of penA in Neisseria meningitidis strains (n = 127), including two nucleotide sites closely associated to reduced susceptibility to penicillins, was amplified and pyrosequenced. All results were in concordance with Sanger sequencing, and a high correlation between alterations in the two Pen(i)-specific sites and reduced susceptibility to penicillins was identified.

Place, publisher, year, edition, pages
Washington, DC: American Society for Microbiology, 2008
Keywords
Anti-Bacterial Agents/pharmacology, Bacterial Proteins/chemistry/genetics, Base Sequence, Humans, Microbial Sensitivity Tests/methods, Molecular Sequence Data, Neisseria meningitidis/*drug effects, Penicillin Resistance, Penicillin-Binding Proteins/chemistry/genetics, Penicillins/*pharmacology, Polymerase Chain Reaction/methods, Sensitivity and Specificity, Sequence Analysis; DNA
National Category
Medical and Health Sciences Infectious Medicine
Research subject
Infectious Diseases; Medicine
Identifiers
urn:nbn:se:oru:diva-3451 (URN)10.1128/AAC.00914-07 (DOI)18070955 (PubMedID)
Available from: 2008-12-08 Created: 2008-12-08 Last updated: 2017-12-14Bibliographically approved
3. Antibiotic susceptibility and characteristics of Neisseria meningitidis isolates from the African meningitis belt, 2000 to 2006: phenotypic and genotypic perspectives
Open this publication in new window or tab >>Antibiotic susceptibility and characteristics of Neisseria meningitidis isolates from the African meningitis belt, 2000 to 2006: phenotypic and genotypic perspectives
Show others...
2009 (English)In: Antimicrobial Agents and Chemotherapy, ISSN 0066-4804, E-ISSN 1098-6596, Vol. 53, no 4, p. 1561-1566Article in journal (Refereed) Published
Abstract [en]

Up-to-date information regarding the antibiotic susceptibility of Neisseria meningitidis strains from African countries is highly limited. Our aim was to comprehensively describe the antibiotic susceptibilities of a selection of N. meningitidis isolates recovered between 2000 and 2006 from 18 African countries, mainly those within the meningitis belt. Susceptibilities to 11 antibiotics were determined using Etest for 137 N. meningitidis isolates (stringently selected from 693 available isolates). The isolates were also characterized by serogrouping, multilocus sequence typing, genosubtyping, and penA allele identification. All N. meningitidis isolates were susceptible to ceftriaxone, chloramphenicol, and ciprofloxacin. No isolate produced beta-lactamase. Only three isolates (2%) displayed reduced susceptibility to penicillin G. The two isolates with the highest penicillin G MICs were the only isolates showing reduced susceptibility to ampicillin and cefuroxime. One of these isolates was also resistant to penicillin V. One percent of isolates displayed reduced susceptibility to rifampin, while 52% of the isolates were resistant to tetracycline, 74% were resistant to erythromycin, and 94% were resistant to sulfadiazine. The MICs of rifampin and tetracycline seemed to be associated with the serogroup of the isolates. In total, 18 sequence types (STs), 10 genosubtypes, and 8 different penA alleles were identified; the most common were ST-7, P1.20,9,35-1, and penA4, respectively. A high level of correlation was found between ST, genosubtype, and penA allele. In conclusion, N. meningitidis isolates from the African meningitis belt remain highly susceptible to the antibiotics used. Regarding beta-lactam antibiotics, rare isolates showed a reduced susceptibility to penicillins, but the expanded-spectrum cephalosporins are not affected at present.

National Category
Medical and Health Sciences Microbiology in the medical area
Research subject
Microbiology; Physiology; Biomedicine
Identifiers
urn:nbn:se:oru:diva-8630 (URN)10.1128/AAC.00994-08 (DOI)19188396 (PubMedID)
Available from: 2009-11-25 Created: 2009-11-23 Last updated: 2018-01-12Bibliographically approved
4. Antibiotic susceptibility of invasive Neisseria meningitidis isolates from 1995 to 2008 in Sweden: the meningococcal population remains susceptible
Open this publication in new window or tab >>Antibiotic susceptibility of invasive Neisseria meningitidis isolates from 1995 to 2008 in Sweden: the meningococcal population remains susceptible
2010 (English)In: Scandinavian Journal of Infectious Diseases, ISSN 0036-5548, E-ISSN 1651-1980, Vol. 42, no 1, p. 61-64Article in journal (Refereed) Published
Abstract [en]

The susceptibility to 7 antibiotics was determined for all Swedish invasive Neisseria meningitidis isolates from 1995 to 2008 (N=717). In general, these remain highly susceptible to the antibiotics recommended for use. Accordingly, penicillin G remains effective for the treatment of invasive meningococcal disease and ciprofloxacin appropriate for prophylaxis.

National Category
Medical and Health Sciences
Research subject
Medicine
Identifiers
urn:nbn:se:oru:diva-8532 (URN)10.3109/00365540903292682 (DOI)000274210500009 ()19883157 (PubMedID)
Available from: 2009-11-25 Created: 2009-11-12 Last updated: 2018-02-23Bibliographically approved
5. Defining the breakpoint for resistance to rifampicin in Neisseria meningitidis by rpoB sequencing
Open this publication in new window or tab >>Defining the breakpoint for resistance to rifampicin in Neisseria meningitidis by rpoB sequencing
Show others...
2009 (English)Manuscript (preprint) (Other academic)
Abstract [en]

Clinical isolates of Neisseria meningitidis resistant to rifampicin are important to identify asthey lead to failure of chemoprophylaxis of meningococcal disease. However, theidentification of these isolates is hindered by the absence of a harmonized breakpoint despiteefforts of standardization. In the present study, a large number (n=352) of clinical N.meningitidis isolates from 12 mainly European countries and spanning over 25 years (1984 to2009) were examined. The collection comprised all clinical isolates with MIC 0.25 mg/lreceived by the national reference laboratories for meningococci in the participating countries(n=161). In addition, representative isolates displaying MIC of rifampicin <0.25 mg/l wereexamined (n=191). Phenotyping and genotyping of isolates were performed and a 660 bpDNA fragment of the rpoB gene was sequenced in all the included isolates. Sequencesdiffering by at least one nucleotide were defined as a unique rpoB allele (n=55). Geometricmeans of MIC were calculated for isolates displaying the same allele. All the clinical isolatesdisplaying MIC >1 mg/l of rifampicin possessed rpoB alleles with critical mutations (in total21 alleles), resulting in substitutions at the codon H552 and less frequently at nearby codons(S548 and S557). These alterations were absent in the alleles (n=34) found in all isolates withMIC 1 mg/l. Based on these findings, rifampicin susceptible isolates could be defined asthose with MIC 1 mg/l. A new web site was created based on the data from this work (http://neisseria.org/nm/typing/rpoB). The rifampicin resistant isolates belonged to diversegenetic lineages and provoked lower bacteremia levels in mice. This biological cost mayexplain the non-expansion of the rifampicin resistant isolates.

National Category
Medical and Health Sciences
Research subject
Medicine
Identifiers
urn:nbn:se:oru:diva-8655 (URN)
Available from: 2009-11-25 Created: 2009-11-25 Last updated: 2017-10-18Bibliographically approved

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Thulin Hedberg, Sara

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