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Phenotypic and molecular characterization of β-lactams resistance in commensal Neisseria strains isolated from neutropenic patients in Tunisia

Phenotypic and molecular characterization of β-lactams resistance in commensal Neisseria strains... Ann Microbiol (2011) 61:695–697 DOI 10.1007/s13213-011-0202-0 SHORT COMMUNICATIONS Phenotypic and molecular characterization of β-lactams resistance in commensal Neisseria strains isolated from neutropenic patients in Tunisia Arij Mechergui & Arabella Touati & Rekaya Baaboura & Wafa Achour & Assia Ben Hassen Received: 16 May 2010 /Accepted: 10 January 2011 /Published online: 12 February 2011 Springer-Verlag and the University of Milan 2011 Abstract We aimed to determine the frequency and the Like other species, commensal Neisseria are naturally molecular basis of β-lactams resistance in a total of 44 transformable and can transfer resistance markers into the commensal Neisseria strains. Bacterial identification was closely related species, Neisseria meningitidis (Maiden performed using standard biochemical tests. Genetic 1998). Thus, they might constitute a potential DNA source diversity of penA gene was studied by penA fingerprint- for the development of antibiotic resistance in meningo- ing. Commensal Neisseria strains were represented essen- cocci precisely in the recent appearance of moderate tially by Neisseria subflava biovar perflava (75%). Four resistance to penicillin in Neisseria meningitidis.In strains (9%) were β-lactamase producers and had the commensal Neisseria species, resistance to penicillin is bla gene. TEM-type β-lactamase was characterized by mainly due to change in the penicillin binding protein TEM bla gene sequencing. PenA fingerprinting gave 32 (PBP) encoded by the penA gene and less frequently to β- TEM patterns with MspI and 19 patterns with TaqI. Our strains lactamase production (Taha and Cavallo 2006). In this presented an important frequency of β-lactamase produc- study, we aimed to determine the prevalence and the tion as well as a high rate of reduced susceptibility to β- molecular basis of β-lactams resistance in commensal lactams with an extensive genetic diversity in the penA Neisseria species. gene polymorphism. . . Keywords Commensal Neisseria β-lactams resistance Materials and methods . . penA gene RFLP β-lactamase From September 2003 to December 2006, a total of 44 commensal Neisseria strains of carriage were collected from Introduction neutropenic patients in the Bone Marrow Transplant Center of Tunisia. Haemophilus influenzae C425 (bla (+)) TEM Commensal Neisseria species are part of the resident (Oueslati et al. 2009)and Neisseria subflava biovar perflava bacterial flora of the human upper respiratory tract and 8022 (R. Leclercq, CHU Caen) were used as quality-control share this ecological niche with Neisseria meningitidis. strains . The identification of commensal Neisseria was based on conventional methods and standard biochemical tests, according to the Janda and Knapp (2003) scheme. : : : : A. Mechergui (*) A. Touati R. Baaboura W. Achour Antimicrobial susceptibility testing was performed as A. Ben Hassen recommended by the CA-SFM guidelines for Neisseria Laboratory unit, Bone Marrow Transplant Center, meningitidis (Soussy 2006). β-lactamase production was Rue Djebel-Lakdhar, Bab-Saadoun, detected by the nitrocefin β-lactamase test (Bio 1006, Tunis, Tunisia e-mail: arij.mechergui@yahoo.fr Mérieux ). For all strains, the penA gene was amplified 696 Ann Microbiol (2011) 61:695–697 Table 1 MIC ranges, MIC values mg/l of antibiotics against Discussion commensal Neisseria strains according to their phenotypic patterns Neisseria subflava biovar perflava predominated (75%). It β-lactams phenotypic MICs (mg/l) patterns is the most frequent commensal Neisseria in adult Penicillin G Amoxicillin Cefotaxim nasopharynx (Perez-Castillo et al. 1994). Of the 44 commensal Neisseria isolates 34% were resistant PS (n=1) 0.032 0.19 0.094 and 64% intermediately resistant to penicillin with MIC of βLP (n=4) 3–32 2–128 0.023–0.125 penicillin G being 0.5 mg/l and 1.5 mg/l, respectively. A βLNRSP MIC range 0.125–8 0.38–2 0.008–2 recent Japanese study indicates that a total of 8.8 and 88.9% (n=39) MIC 1 1 0.064 of 45 isolates of Neisseria subflava were resistant and intermediately resistant to penicillin G, respectively, with PS penicillin susceptible, βLP β-lactamase positive, βLNRSP β - lactamase negative with reduced susceptibility to penicillin MIC and MIC of penicillin being 0.5 mg/l and 1 mg/l, 50 90 respectively (Furuya et al. 2007). In our study, amoxicillin- resistant strains represented 9% of all strains. They represent by PCR with PFup1 and GCdown3 primers (Perez- 0% in Neisseria meningitidis in Tunisia (Saquer et al. 2006). Castillo et al. 1994) and digested with MspIand TaqI No resistance to cefotaxim was observed in our study and (Promega) (Zhang et al. 1990). For β-lactamase positive MIC of this antibiotic was of 0.064 mg/l. In Neisseria strains, bla gene was amplified (Dabernat et al. 2002) TEM meningitidis; third generation cephalosporin represents the and sequenced using ABI PRISM 377 DNA (Applied least modified antibiotic and preserve low MICs compatible Biosystems). with therapeutic use (Cavallo 2006). PenA gene fingerprinting of amplified DNA provided higher resolution by the use of MspI, which provided Results approximately 2–8 DNA fragments from the gene, com- pared with TaqI, which produced only about 2–5 fragments. Commensal Neisseria isolates were represented essentially The same result was found in Neisseria meningitidis by Neisseria subflava biovar perflava (75%) followed by N. (Zhang et al. 1990). The extensive diversity found in the mucosa (14%), N. sicca (9%) and N. polysaccharea (2%). penA gene from penicillin-resistant meningococci could Of the 44 isolates, 98% (34% resistant and 64% interme- thus be a consequence of the creation, on multiple diately resistant) had reduced susceptibility to penicillin G, occasions, of mosaic genes. Further diversity is probably 57% to amoxicillin (9% resistant and 48% intermediately generated by truncation of the ends of the regions resistant), and 0% to cefotaxim. Four strains (9%) were β- introduced from commensal species, and perhaps by lactamase positive (βLP), 39 strains (89%) were β- mismatch repair processes, as the original mosaic penA lactamase negative with reduced susceptibility to penicillin genes have spread horizontally through the meningococcal (βLNRSP), and 1 strain (2%) was susceptible to penicillin population (Zhang et al. 1990). (PS) (Table 1). In our study, the four β-lactamase positive strains (9%) RFLP revealed respectively 19 and 32 different TaqI and harboured bla 1 gene. Indeed, TEM-1 enzyme has been TEM MspI digest patterns among the 44 commensal Neisseria found frequently in Neisseria species. It is also the most isolates (Fig. 1). frequent type widespread in enterobacteria. However, in The four βLP strains had positive results with bla Neisseria meningitidis, β-lactamase production is excep- TEM gene PCR amplification. Sequence analysis showed that tional and has been observed in only a few countries those strains contained a TEM-1 β-lactamase gene. (Canada, Spain and South Africa) (Cavallo 2006). Fig. 1 PCR restriction fragment-length polymorphism analysis patterns of penA amplicons digested with TaqI (a) and MspI(b) for some clinical strains M 100-pb size marker; lanes 1–7 commensal Neisseria strains with reduced susceptibility to β-lactams Ann Microbiol (2011) 61:695–697 697 Acknowledgments We gratefully acknowledge Pr. R. Leclercq and Maiden MCJ (1998) Horizontal genetic exchange, evolution, and spread Pr A. Kechrid for supplying us the reference strains used in this study. of antibiotic resistance in bacteria. Clin Infect Dis 27(1):12–20 Oueslati S, Smaoui H, Joubert G, Dabernat H, Kechrid A (2009) Etude de la resistance aux β-lactamines et des marqueurs moleculaires chez 157 souches d'Haemophilus influenzae isolées References chez l'enfant à Tunis. Can J Microbiol 55(5):515–519 Perez-Castillo A, Perez-Castillo AM, Saez-Nieto JA (1994) Sequence Cavallo JD (2006) Neisseria gonorrhoeae. In: Courvalin P, of the penicillin–binding protein 2-encoding gene (penA)of Leclercq R, Bingen E (eds) Antibiogramme. ESKA, France, Neisseria perflava/sicca. Gene 146:91–93 pp 429–435 Saquer A, Smaoui H, Kechrid A (2006) Typage phénotypique et étude Dabernat H, Delmas C, Seguy M, Pelissier R, Faucon G, Bennamani de la sensibilité aux antibiotiques chez Neisseria meningitidis S, Pasquier C (2002) Diversity of β-lactam resistance conferring isolées à l’hôpital d’enfants de Tunis. Tunis Méd 24:730–733 amino acid substitutions in penicillin–binding protein 3 of Soussy CJ (2006) Comité de l’antibiogramme de la société française Haemophilus influenzae. Antimicrob Agents Chemother 46 de microbiologie. (7):2208–2218 Taha MK, Cavallo JD (2006) Neisseria meningitidis. In: Courvalin P, Furuya R, Onoye Y, Kanayama A, Saika T, Iyoda T, Tatewaki M, Leclercq R, Bingen E (eds) Antibiogramme. ESKA, France, pp Matsuzaki I, Tanaka M (2007) Antimicrobial resistance in 419–427 clinical isolates of Neisseria subflava from the oral cavities of a Zhang QY, Jones DM, Saez-Nieto JA, Trallero EM, Spratt BG (1990) japanese population. J Infect Chemother 13(5):302–304 Genetic diversity of penicillin–binding protein 2 genes of Janda WM, Knapp JS (2003) Neisseria and Morexella catarrhalis. In: penicillin resistant strains of Neisseria meningitidis revealed by Murray PR et al (eds) Manual of clinical microbiology. ASM fingerprinting of amplified DNA. Antimicrob Agents Chemother Press, Washington, pp 585–608 34(8):1523–1528 http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Annals of Microbiology Springer Journals

Phenotypic and molecular characterization of β-lactams resistance in commensal Neisseria strains isolated from neutropenic patients in Tunisia

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Publisher
Springer Journals
Copyright
Copyright © 2011 by Springer-Verlag and the University of Milan
Subject
Life Sciences; Microbiology; Microbial Genetics and Genomics; Microbial Ecology; Mycology; Medical Microbiology; Applied Microbiology
ISSN
1590-4261
eISSN
1869-2044
DOI
10.1007/s13213-011-0202-0
Publisher site
See Article on Publisher Site

Abstract

Ann Microbiol (2011) 61:695–697 DOI 10.1007/s13213-011-0202-0 SHORT COMMUNICATIONS Phenotypic and molecular characterization of β-lactams resistance in commensal Neisseria strains isolated from neutropenic patients in Tunisia Arij Mechergui & Arabella Touati & Rekaya Baaboura & Wafa Achour & Assia Ben Hassen Received: 16 May 2010 /Accepted: 10 January 2011 /Published online: 12 February 2011 Springer-Verlag and the University of Milan 2011 Abstract We aimed to determine the frequency and the Like other species, commensal Neisseria are naturally molecular basis of β-lactams resistance in a total of 44 transformable and can transfer resistance markers into the commensal Neisseria strains. Bacterial identification was closely related species, Neisseria meningitidis (Maiden performed using standard biochemical tests. Genetic 1998). Thus, they might constitute a potential DNA source diversity of penA gene was studied by penA fingerprint- for the development of antibiotic resistance in meningo- ing. Commensal Neisseria strains were represented essen- cocci precisely in the recent appearance of moderate tially by Neisseria subflava biovar perflava (75%). Four resistance to penicillin in Neisseria meningitidis.In strains (9%) were β-lactamase producers and had the commensal Neisseria species, resistance to penicillin is bla gene. TEM-type β-lactamase was characterized by mainly due to change in the penicillin binding protein TEM bla gene sequencing. PenA fingerprinting gave 32 (PBP) encoded by the penA gene and less frequently to β- TEM patterns with MspI and 19 patterns with TaqI. Our strains lactamase production (Taha and Cavallo 2006). In this presented an important frequency of β-lactamase produc- study, we aimed to determine the prevalence and the tion as well as a high rate of reduced susceptibility to β- molecular basis of β-lactams resistance in commensal lactams with an extensive genetic diversity in the penA Neisseria species. gene polymorphism. . . Keywords Commensal Neisseria β-lactams resistance Materials and methods . . penA gene RFLP β-lactamase From September 2003 to December 2006, a total of 44 commensal Neisseria strains of carriage were collected from Introduction neutropenic patients in the Bone Marrow Transplant Center of Tunisia. Haemophilus influenzae C425 (bla (+)) TEM Commensal Neisseria species are part of the resident (Oueslati et al. 2009)and Neisseria subflava biovar perflava bacterial flora of the human upper respiratory tract and 8022 (R. Leclercq, CHU Caen) were used as quality-control share this ecological niche with Neisseria meningitidis. strains . The identification of commensal Neisseria was based on conventional methods and standard biochemical tests, according to the Janda and Knapp (2003) scheme. : : : : A. Mechergui (*) A. Touati R. Baaboura W. Achour Antimicrobial susceptibility testing was performed as A. Ben Hassen recommended by the CA-SFM guidelines for Neisseria Laboratory unit, Bone Marrow Transplant Center, meningitidis (Soussy 2006). β-lactamase production was Rue Djebel-Lakdhar, Bab-Saadoun, detected by the nitrocefin β-lactamase test (Bio 1006, Tunis, Tunisia e-mail: arij.mechergui@yahoo.fr Mérieux ). For all strains, the penA gene was amplified 696 Ann Microbiol (2011) 61:695–697 Table 1 MIC ranges, MIC values mg/l of antibiotics against Discussion commensal Neisseria strains according to their phenotypic patterns Neisseria subflava biovar perflava predominated (75%). It β-lactams phenotypic MICs (mg/l) patterns is the most frequent commensal Neisseria in adult Penicillin G Amoxicillin Cefotaxim nasopharynx (Perez-Castillo et al. 1994). Of the 44 commensal Neisseria isolates 34% were resistant PS (n=1) 0.032 0.19 0.094 and 64% intermediately resistant to penicillin with MIC of βLP (n=4) 3–32 2–128 0.023–0.125 penicillin G being 0.5 mg/l and 1.5 mg/l, respectively. A βLNRSP MIC range 0.125–8 0.38–2 0.008–2 recent Japanese study indicates that a total of 8.8 and 88.9% (n=39) MIC 1 1 0.064 of 45 isolates of Neisseria subflava were resistant and intermediately resistant to penicillin G, respectively, with PS penicillin susceptible, βLP β-lactamase positive, βLNRSP β - lactamase negative with reduced susceptibility to penicillin MIC and MIC of penicillin being 0.5 mg/l and 1 mg/l, 50 90 respectively (Furuya et al. 2007). In our study, amoxicillin- resistant strains represented 9% of all strains. They represent by PCR with PFup1 and GCdown3 primers (Perez- 0% in Neisseria meningitidis in Tunisia (Saquer et al. 2006). Castillo et al. 1994) and digested with MspIand TaqI No resistance to cefotaxim was observed in our study and (Promega) (Zhang et al. 1990). For β-lactamase positive MIC of this antibiotic was of 0.064 mg/l. In Neisseria strains, bla gene was amplified (Dabernat et al. 2002) TEM meningitidis; third generation cephalosporin represents the and sequenced using ABI PRISM 377 DNA (Applied least modified antibiotic and preserve low MICs compatible Biosystems). with therapeutic use (Cavallo 2006). PenA gene fingerprinting of amplified DNA provided higher resolution by the use of MspI, which provided Results approximately 2–8 DNA fragments from the gene, com- pared with TaqI, which produced only about 2–5 fragments. Commensal Neisseria isolates were represented essentially The same result was found in Neisseria meningitidis by Neisseria subflava biovar perflava (75%) followed by N. (Zhang et al. 1990). The extensive diversity found in the mucosa (14%), N. sicca (9%) and N. polysaccharea (2%). penA gene from penicillin-resistant meningococci could Of the 44 isolates, 98% (34% resistant and 64% interme- thus be a consequence of the creation, on multiple diately resistant) had reduced susceptibility to penicillin G, occasions, of mosaic genes. Further diversity is probably 57% to amoxicillin (9% resistant and 48% intermediately generated by truncation of the ends of the regions resistant), and 0% to cefotaxim. Four strains (9%) were β- introduced from commensal species, and perhaps by lactamase positive (βLP), 39 strains (89%) were β- mismatch repair processes, as the original mosaic penA lactamase negative with reduced susceptibility to penicillin genes have spread horizontally through the meningococcal (βLNRSP), and 1 strain (2%) was susceptible to penicillin population (Zhang et al. 1990). (PS) (Table 1). In our study, the four β-lactamase positive strains (9%) RFLP revealed respectively 19 and 32 different TaqI and harboured bla 1 gene. Indeed, TEM-1 enzyme has been TEM MspI digest patterns among the 44 commensal Neisseria found frequently in Neisseria species. It is also the most isolates (Fig. 1). frequent type widespread in enterobacteria. However, in The four βLP strains had positive results with bla Neisseria meningitidis, β-lactamase production is excep- TEM gene PCR amplification. Sequence analysis showed that tional and has been observed in only a few countries those strains contained a TEM-1 β-lactamase gene. (Canada, Spain and South Africa) (Cavallo 2006). Fig. 1 PCR restriction fragment-length polymorphism analysis patterns of penA amplicons digested with TaqI (a) and MspI(b) for some clinical strains M 100-pb size marker; lanes 1–7 commensal Neisseria strains with reduced susceptibility to β-lactams Ann Microbiol (2011) 61:695–697 697 Acknowledgments We gratefully acknowledge Pr. R. Leclercq and Maiden MCJ (1998) Horizontal genetic exchange, evolution, and spread Pr A. Kechrid for supplying us the reference strains used in this study. of antibiotic resistance in bacteria. Clin Infect Dis 27(1):12–20 Oueslati S, Smaoui H, Joubert G, Dabernat H, Kechrid A (2009) Etude de la resistance aux β-lactamines et des marqueurs moleculaires chez 157 souches d'Haemophilus influenzae isolées References chez l'enfant à Tunis. Can J Microbiol 55(5):515–519 Perez-Castillo A, Perez-Castillo AM, Saez-Nieto JA (1994) Sequence Cavallo JD (2006) Neisseria gonorrhoeae. In: Courvalin P, of the penicillin–binding protein 2-encoding gene (penA)of Leclercq R, Bingen E (eds) Antibiogramme. ESKA, France, Neisseria perflava/sicca. Gene 146:91–93 pp 429–435 Saquer A, Smaoui H, Kechrid A (2006) Typage phénotypique et étude Dabernat H, Delmas C, Seguy M, Pelissier R, Faucon G, Bennamani de la sensibilité aux antibiotiques chez Neisseria meningitidis S, Pasquier C (2002) Diversity of β-lactam resistance conferring isolées à l’hôpital d’enfants de Tunis. Tunis Méd 24:730–733 amino acid substitutions in penicillin–binding protein 3 of Soussy CJ (2006) Comité de l’antibiogramme de la société française Haemophilus influenzae. Antimicrob Agents Chemother 46 de microbiologie. (7):2208–2218 Taha MK, Cavallo JD (2006) Neisseria meningitidis. In: Courvalin P, Furuya R, Onoye Y, Kanayama A, Saika T, Iyoda T, Tatewaki M, Leclercq R, Bingen E (eds) Antibiogramme. ESKA, France, pp Matsuzaki I, Tanaka M (2007) Antimicrobial resistance in 419–427 clinical isolates of Neisseria subflava from the oral cavities of a Zhang QY, Jones DM, Saez-Nieto JA, Trallero EM, Spratt BG (1990) japanese population. J Infect Chemother 13(5):302–304 Genetic diversity of penicillin–binding protein 2 genes of Janda WM, Knapp JS (2003) Neisseria and Morexella catarrhalis. In: penicillin resistant strains of Neisseria meningitidis revealed by Murray PR et al (eds) Manual of clinical microbiology. ASM fingerprinting of amplified DNA. Antimicrob Agents Chemother Press, Washington, pp 585–608 34(8):1523–1528

Journal

Annals of MicrobiologySpringer Journals

Published: Feb 12, 2011

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