In a collection of 110 clinical isolates of Klebsiella pneumoniae, a single strain, Kp593, was found to exhibit a mutator phenotype with a rifampicin mutation frequency 100-fold higher than the modal value for this species. Complementation experiments with the wild-type MutL, one of the main components of the methyl-directed mismatch repair system, allowed the mutator phenotype to be reversed. Sequencing revealed substitution of the conserved residue Lys307 to Arg and site-directed mutagenesis followed by complementation experiments confirmed the critical role of this mutation. The patient infected with Kp593 relapsed a month later and the strain isolated then, Kp869, was identical to Kp593, as verified by PFGE analysis. Phenotypically, Kp869 colonies were more mucoid than those of Kp593, probably due to increased capsule synthesis as shown by electron microscopy. In addition, Kp869 exhibited a 16-fold higher amoxicillin resistance level related to a 36.4 kb tandem duplication encompassing the chromosomal blaSHV-11 gene, which was unstable in vitro. These data suggest that the mutator phenotype found in Kp593/Kp869 is associated with beneficial mutations conferring a selective advantage, such as increased virulence factor production and antibiotic resistance. The latter was due to resistance gene duplication, an event rarely described in natural isolates. This is the first description of the in vivo occurrence of gene duplication in a mutator background.
BrochetM.,
CouveE.,
ZouineM.,
PoyartC.,
GlaserP.2008; A naturally occurring gene amplification leading to sulfonamide and trimethoprim resistance in Streptococcus agalactiae . J Bacteriol 190:672–680
De ChampsC.,
RichC.,
ChandezonP.,
ChanalC.,
SirotD.,
ForestierC.2004; Factors associated with antimicrobial resistance among clinical isolates of Klebsiella pneumoniae : 1-year survey in a French university hospital. Eur J Clin Microbiol Infect Dis 23:456–462
DenamurE.,
BonacorsiS.,
GiraudA.,
DuriezP.,
HilaliF.,
AmorinC.,
BingenE.,
AndremontA.,
PicardB.other authors2002; High frequency of mutator strains among human uropathogenic Escherichia coli isolates. J Bacteriol 184:605–609
FischerI.,
GrobostF.,
DutilhB.,
CoutureJ. F.,
JullinJ.,
FourmauxS.,
DucastaingA.,
LagrangeI.,
NouryP.other authors2004; Antibiotic resistance among isolates of Enterobacteriaceae and Acinotobacter baumannii in health care centers served by private laboratories: a 6-month study in the Aquitaine area. In 6th European Congress on Chemotherapy and Infection & 24th Interdisciplinary Anti-Infectious Chemotherapy Meeting Paris1–3 December 2004 Abstract no. 413/81P
GuarnéA., Ramon-MaiquesS.,
WolffE. M.,
GhirlandoR.,
HuX.,
MillerJ. H.,
YangW.2004; Structure of the MutL C-terminal domain: a model of intact MutL and its roles in mismatch repair. EMBO J 23:4134–4145
HaeggmanS.,
LofdahlS.,
PaauwA.,
VerhoefJ.,
BrisseS.2004; Diversity and evolution of the class A chromosomal β -lactamase gene in Klebsiella pneumoniae . Antimicrob Agents Chemother 48:2400–2408
HoangT. T.,
Karkhoff-SchweizerR. R.,
KutchmaA. J.,
SchweizerH. P.1998; A broad-host-range Flp-FRT recombination system for site-specific excision of chromosomally-located DNA sequences: application for isolation of unmarked Pseudomonas aeruginosa mutants. Gene 212:77–86
JunopM. S.,
YangW.,
FunchainP.,
ClendeninW.,
MillerJ. H.2003; In vitro and in vivo studies of MutS, MutL and MutH mutants: correlation of mismatch repair and DNA recombination. DNA Repair (Amst 2:387–405
MathewA.,
HarrisA. M.,
MarshallM. J.,
RossG. W.1975; The use of analytical isoelectric focusing for detection and identification of β -lactamases. J Gen Microbiol 88:169–178
Members of the SFM Antibiogram Committee2003; Comité de l'Antibiogramme de la Société Francaise de Microbiologie Report 2003. Int J Antimicrob Agents 21:364–391
OliverA.,
CantonR.,
CampoP.,
BaqueroF.,
BlazquezJ.2000; High frequency of hypermutable Pseudomonas aeruginosa in cystic fibrosis lung infection. Science 288:1251–1254
PrunierA. L.,
MalbrunyB.,
LauransM.,
BrouardJ.,
DuhamelJ. F.,
LeclercqR.2003; High rate of macrolide resistance in Staphylococcus aureus strains from patients with cystic fibrosis reveals high proportions of hypermutable strains. J Infect Dis 187:1709–1716
RiceL. B.,
CariasL. L.,
HujerA. M.,
BonafedeM.,
HuttonR.,
HoyenC.,
BonomoR. A.2000; High-level expression of chromosomally encoded SHV-1 β -lactamase and an outer membrane protein change confer resistance to ceftazidime and piperacillin-tazobactam in a clinical isolate of Klebsiella pneumoniae . Antimicrob Agents Chemother 44:362–367
WatsonM. E.Jr,
BurnsJ. L.,
SmithA. L.2004; Hypermutable Haemophilus influenzae with mutations in mutS are found in cystic fibrosis sputum. Microbiology 150:2947–2958
WorthL.Jr,
ClarkS.,
RadmanM.,
ModrichP.1994; Mismatch repair proteins MutS and MutL inhibit RecA-catalyzed strand transfer between diverged DNAs. Proc Natl Acad Sci U S A 91:3238–3241