1. EPIDEMIOLOGY AND INFECTION CONTROL OF ESBL-PRODUCING K
EPIDEMIOLOGY AND INFECTION CONTROL OF ESBL-PRODUCING K. PNEUMONIAE STRAINS CAUSED 5 OUTBREAKS IN A HUNGARIAN NICU DURING THE YEARS
Damjanova, I¹., Z. Ozsvár², Á. Tóth¹, I. Csanádiné Petró², L. Szikra², J. Pászti¹, G. Simon²
¹National Center for Epidemiology, Budapest, Hungary
²„Szt. Gyórgy” Hospital, Székesfehérvár, Hungary
ABSTRACT
RESULTS
Objectives: The aim of this study was to perform a prospective survey on prevalence and epidemiological relatedness among ESBL-producing K. pneumoniae (ESBL-KP) strains isolated in a Hungarian NICU during a four-year period.
Methods: All patients admitted to NICU during the four-year period were screened for colonisation with ESBL-producing K. pneumoniae. Confirmation of ESBL production was done by E-test, double-disc diffusion and agar dilution methods according to NCCLS. We performed PCR analysis for beta-lactamase detection using specific primers; phage typing, resistance transfer, plasmid profile analysis and plasmid REA, genomic fingerprinting by ERIC-PCR and PFGE to determine the genetic relatedness among strains.
Results: Among 210 K. pneumoniae isolates recovered from NICU between , 119 were ESBL-producers but non of these was isolated in ESBL isolates were recovered from five outbreak periods. The outbreak strains showed multidrug-resistance with similar antibiotypes. 87 of 107 isolates, belonged to six different phage types, but 20 were not typable. PFGE analysis revealed six different genetic clones at 90% similarity level. .All isolates harboured plasmids ranging from 2,6 to 203 kb and 14 different plasmid profiles were identified. The same plasmid of 94 kb was obtained from the transconjugants of six outbreak strains by plasmid restriction analysis The sequence analysis of the SHV PCR products showed that SHV-5 was presented in all outbreak clones and their transconjugants.
Conclusions: This prospective study confirms that the same ESBL coding plasmid persisted from 2001 to 2004 in this hospital ward, while five different outbreak clones were revealed during the given period. After the change of the antibiotic treatment regiment at the beginning of 2004 there were no more ESBL-producers isolated.
The complex typing results of 107 K. pneumoniae strains isolated from 5 different outbreaks during 4 years were summarized in tables 1and 2.
Antibiotic resistance phenotypes: According to the disk tests all off isolates were resistant to ceftazidime, cefotaxime, mostly to ampicillin/sulbactam, tazobactam, netilmicin, rarely to cefepime and trimethoprime and susceptible to ciprofloxacin.
Phage typing: The examinated strains were classified into 6 different phage types, but 25% of them were not typeable by phages. In general the strains from different outbreaks belonged to different phage types.
ERIC-PCR typing: The 107 K. pneumoniae isolates were ranged in 4 ERIC-subtypes. Isolates from 3 different outbreaks showed very similar ERIC-patterns .
PFGE typing: The isolates subjected to PFGE gave 6 clusters (pulso types) at 90% similarity level. Each cluster was interpreted as group of clonally related isolates and it was found that in NICU they were isolated 5 different epidemic clones and one additional sporadic clone, unrelated to each other.
Plasmid profile analysis: 14 distinct plasmid profiles were obtained from 107 isolates. 101 of them harboured plasmid of approx. 94 kb in size.
Sequencing of β-lactamase genes: SHV-5 was presented in the representative isolates from each of the 5 examined outbreaks.
Transfer of resistance determinants of 8 representative isolates derived from 5 different outbreaks was carried out. From all donors only one plasmid of approximately 94 kb was transferred. Plasmids from 8 transconjugants were digested with EcoRI (Fig. 2) and identical fingerprints were obtained.
By PCR amplification it was proved that this plasmid harbored the SHV-5 encoding gene.
M K K M
48,5 kb
FIG. 2. EcoRI fingerprint of plasmids isolated from transconjugants (TK) of 4 outbreak-representative isolates ones. Lanes: M: molecular weight marker; 1: TK36/02, clone D; 2: TK39/02, clone E; 3: TK85/02, clone E; 4: TK109/03, clone J;
Fig. 1. PFGE-XbaI profiles of different epidemic clones. Lanes: M: molecular weight marker (lambda DNA ladder, Bio-Rad Laboratories, CA, USA),); lanes K: ATCC ; lane 1: K239/04,clone O, Outbreak (OB) I; lane 2: K234/04, clone E, OB I; lane 3: K231/04, clone O, OB I; lane 4: K19/02, clone P, OB II; lane 5: K21/02, clone E, OB II; lane 6: K41/02, clone E, OB II; lane 7: K47/02, clone D, OB II; lane 8: K80/02, clone E, OB II; lane 9: K81/02, clone E, OB II; lane 10: K93/02, clone E, OB III; lane 11: K109/03, clone J, OB IV; lane 12: K112/03, clone J, OB IV; lane 13: K133/03, clone J, OB IV; lane 14: K217/03, clone M, OB V; lane 15: K231/03, clone M, OB V;
INTRODUCTION AND PURPOSE 
ESBL producing Klebsiella spp. are world–wide the most common multiple resistant Gram-negative bacteria (1). The international survey examining Klebsiellae from intensive care units in Western and Southern Europe proved 1-59% ESBLs from K. pneumoniae and K. oxytoca (2).
In Hungary the occurrence of ESBL-producing Klebsiella pneumoniae has been poorly described previously: in 1998 they were isolated from sporadic cases (3) and in the same year an SHV–5 type ESBL–producing K. pneumoniae clone caused outbreak in a perinatal intensive care unit in a county hospital (4). Our systematical examinations on nosocomial infections draw our attention on the ESBL-producing Klebsiellae infections occurred in different hospitals in Hungary. From 1998 to 2003 there were 25 referred nosocomial outbreaks in Hungarian NICUs, among these 19 were caused by Klebsiellae. The aim of present work was (i) to delineate the clonal identity or diversity of the ESBL-producing Klebsiella spp. strains caused outbreaks in the same NICU during four years, (ii) to determine the persistance and dissemination of a single clone or different, possibly variable clones in the same hospital ward, (iii) to establish the frequent ESBL types among outbreak-causing K. pneumoniae strains and (iv) to determine the major risk factors for ESBL-KP infection.
Table 2. Antimicrobial MIC values of ESBL-producing K. pneumoniae outbreak-representative isolates and their transconjugants
MATERIALS and METHODS
Setting: The study was carried out in a 110-bed pediatric ward in „Szt Gyórgy” county hospital. The neonatal intensive care unit (NICU) has 23 bed. After the first isolation of ESBL-KP in this NICU in 2001 continous screening was performed
Bacterial strains: 119 ESBL-producing K. pneumoniae strains were recovered from NICU between of them were isolated from 5 outbreak periods and from 67 patients.
Biochemical identification of the isolates was carried out by using the ATB ID 32 E (Bio Mérieux).
Susceptibility testing: Preliminary antibiotic susceptibility tests were performed by using the Kirby-Bauer disk diffusion method in the clinical laboratories following NCCLS guidelines(5). The putative production of an ESBL was detected by combined disk method ESBL SET (Mast Diagnostics).
The MICs of representativ strains and its transconjgants were determined by E-test to ceftazidime, cefotaxime, ceftriaxone, cefepime, amoxacillin/clavulanic acid, gentamicin, tobramycin, netilmicin, amikacin and ciprofloxacin.
Phage typing was carried out according to Slopek et al (6) with 19 phages.
Plasmid DNA analysis: Plasmid DNA was obtained from 107 isolates and from 8 transconjugants by alakaline lysis method (7) and analysed by electrophoresis on 0.7% agarose gels. For plasmid fingerprinting plasmid DNA from transconjugants was digested with EcoRI and electrophoresed in 0.9% agarose at 100 V for 2.5 h.
Transfer of resistance determinants: The mating experiments were carried out with 8 ESBL-producing outbreak representative strains and the transconjugants were selected on agar containing rifampin (300mg/l) and cefotaxime (4.0mg/l)
ERIC-PCR typing: ERIC-PCR was carried out on all isolates according to Welsh and Mc Clelland (8) using ERIC-2 primer. .
PCR amplification of blasSHV genes. The general SHV primers were used for synthesis of the 1016 bp amplicon. Amplification of blashv gene was carried out with all isolates and transconjgants.
SHV-RFLP analysis of the amplified blaSHV genes with NheI endonuclease was used to identify glycine to serine substitution at position 238 (9).
DNA sequencing: DNA sequensing was performed using ABI prism TM Big DyeTM Terminator Cycle Sequencing Ready Reaction Kit with Ampli TaqR DNA Polymerase with FS Perkin-Elmer product. The cleaned samples were analysed in a 310 Genetic Analyser ABI PrismTM.
Pulsed field gel electrophoresis method was based on the standardized CDC protocols (10). DNA-containing agar blocks were digested with 50U XbaI for 16 h and XbaI fragments were separated with ramped pulses of 2,16 to 54,17 s for 22 h. The gels were visualised with the Gel-Doc system (Bio-Rad) and analysed by Fingerprinting 2 software package (Bio-Rad) according to the criteria of Tenover et al. (11)
Table 1. Complex typing results of 107 ESBL-KP strains isolated from outbreaks between
CONCLUSIONS
During the four year study period 5 epidemic clones caused 5 outbreaks in the same NICU;
The 107 ESBL-KP isolates were grouped in 6 phage types and not typable group, 4 ERIC-PCR subtypes and 6 pulso types;
There was evidence that one of the epidemic clones isolated from 3 different outbreaks persisted for 15 months in the same hospital ward;
Dissemination of an 94 kb epidemic plasmid was responsible for SHV-5 encoding gene acquisition by different K. pneumoniae strains and makes them able to produce outbreaks;
The major risks for ESBL-KP infection in this NICU are the profilactic antibiotic therapy by aminoglycosids and oxyimino-cephalosporins and the use of intravenous line hubs.
Restricted use of oxyimino-β-lactams and aminoglycosides was initiated in September In addition after reinstatement of strict handwashing and thorough environmental cleaning in 2004 there were no more ESBL-producers isolated from this NICU.
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a: non typable; b1,2,3: number of strains isolated from blood cultures