JAC Advance Access originally published online on March 10, 2006
Journal of Antimicrobial Chemotherapy 2006 57(5):979-982; doi:10.1093/jac/dkl077
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Molecular epidemiology of extended-spectrum ß-lactamase-producing Klebsiella pneumoniae in a neonatal intensive care unit
1 Dipartimento di Scienze Mediche Preventive, Sezione di Igiene, Università di Napoli ‘Federico II’, Napoli, Italy; 2 Dipartimento di Scienze per la Salute, Università del Molise, Campobasso, Italy; 3 Dipartimento di Pediatria, Università di Napoli ‘Federico II’, Napoli, Italy; 4 Dipartimento di Medicina Sperimentale e Patologia, Università di Roma ‘La Sapienza’, Roma, Italy; 5 CEINGE Biotecnologie Avanzate, Napoli, Italy
* Corresponding author. Tel: +39-081-7463026; Fax: +39-081-7463352; E-mail: rafzarri{at}unina.it
Received 9 November 2005; returned 26 January 2006; revised 15 February 2006; accepted 16 February 2006
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Abstract |
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Objectives: To investigate the molecular epidemiology of extended-spectrum ß-lactamase (ESBL)-producing Klebsiella pneumoniae in the neonatal intensive care unit of a university hospital in Italy.
Methods: Antibiotic susceptibility was evaluated by disc diffusion and Etest. ESBLs were identified by isoelectric focusing, PCR and DNA sequencing analysis. Genotyping was performed by PFGE analysis. Conjugation was performed by broth mating.
Results: Molecular typing of K. pneumoniae isolates identified three distinct PFGE patterns. Isolates of PFGE profile A were isolated during an epidemic in 1996, while isolates of PFGE profiles B and C were sequentially isolated from September 2002 to December 2004, when 233 colonizations and 19 infections by K. pneumoniae occurred. All K. pneumoniae strains of different PFGE types were identified as ESBL producers. DNA sequencing of amplified ß-lactamase genes identified a novel blaTEM ESBL (blaTEM-136) along with blaSHV-1 in chromosomal and plasmid DNA from K. pneumoniae of PFGE type A, respectively, and blaTEM-1 and blaSHV-12 in plasmid DNA from K. pneumoniae of PFGE types B and C. Conjugation experiments demonstrated that resistance to third-generation cephalosporins, along with an 
80 kb plasmid containing blaSHV-12 and blaTEM-1, was transferred from K. pneumoniae epidemic strains of PFGE types B and C to a susceptible Escherichia coli host at a frequency of 4 x 10–6 and 1 x 10–6 cfu/recipient cell, respectively.
Conclusions: The selection of ESBL-producing clones and the transfer of the blaSHV-12 ESBL gene between different clones were responsible for the spread of K. pneumoniae in the neonatal intensive care unit.
Keywords: nosocomial infections , antimicrobial resistance , genotyping , horizontal gene transfer
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Introduction |
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Klebsiella pneumoniae producing extended-spectrum ß-lactamases (ESBLs) has been increasingly implicated in nosocomial outbreaks in neonatal intensive care units (NICUs).1–4 ESBL-producing K. pneumoniae, which caused an outbreak during 1996 in the NICU of the ‘Federico II’ University Hospital of Naples, Italy,4 was not isolated in the ward again until August 2002. After that, a new outbreak occurred, lasting until December 2004. The objectives of the present study were to investigate the molecular epidemiology of ESBL-producing K. pneumoniae colonization and infection in the NICU of our university hospital and to study the spread of ESBLs in different K. pneumoniae strains.
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Materials and methods |
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The NICU of the 1200-bed teaching hospital of the University ‘Federico II’ of Naples consists of three rooms with a maximum capacity of eight neonates per room. Washing sinks are available in each room and gloves are used routinely. Nosocomial infection surveillance in the NICU was performed as previously described.4 In brief, surveillance swabs from the nose, pharynx and rectum of all neonates admitted to the ward were collected on a weekly basis and environmental investigations were also performed. Written informed consent to participate in this study was obtained from patients' parents.
All ESBL-producing K. pneumoniae strains isolated from surveillance swabs, from clinical samples (blood culture, urine, bronchial aspirate, ocular swab) and from environmental samples during September 2002–December 2004 were studied. Two ESBL-producing K. pneumoniae strains isolated during 1996 in the same ward4 were also analysed. Escherichia coli ATCC 25922 was used as an ESBL-negative reference strain, and K. pneumoniae ATCC 700603 was used as an ESBL-positive reference strain.
Susceptibilities to antimicrobial agents were determined by the antibiotic disc diffusion method.5 ESBL activity was first evaluated using the standard disc diffusion test for cephalosporins and monobactam, then using the double-disc synergy test between cephalosporins or monobactam and clavulanate.5 ESBL activity was confirmed by Etest cefotaxime/cefotaxime + clavulanic and ceftazidime/ceftazidime + clavulanic acid strips (AB BIODISK, Solna, Sweden) as recommended by the manufacturer. Broth mating was performed as previously described.6 The mating rate was calculated as the frequency of ceftriaxone-resistant transconjugants per recipient cell resistant to sodium azide after 18 h of selection.
Isoelectric focusing (IEF) was performed using precast Ampholine® PAGplates pH 3.5–9.5 (Amersham BioSciences, Cologno Monzese, Milan, Italy) following the instructions provided by the supplier.
DNA macrorestriction of K. pneumoniae isolates, PFGE analysis and interpretation of the resulting restriction patterns were performed as previously described.4
DNA purifications were performed using the Wizard® Genomic DNA purification kit and the Wizard® Plus SV minipreps DNA purification system adapted for low-copy-number plasmids (Promega Corporation, Madison, WI, USA) according to the manufacturer's procedure. PCR amplifications for SHV, TEM and CTX genes were performed as previously described.7,8 Direct DNA sequencing of gel-purified PCR products was performed using the ABI PRISMR BigDyeTM Terminator v3.0 Ready Reaction Cycle Sequencing Kit and the Applied Biosystems 3100 Genetic Analyzer (Applied Biosystems, Foster City, CA, USA) as recommended by the manufacturer.
The nucleotide sequences of SHV-1 and TEM-136 ß-lactamases from K. pneumoniae strain 403 and SHV-12 ß-lactamase from K. pneumoniae strain 2183 have been deposited in the GenBank nucleotide database under accession numbers AY826416 [GenBank] , AY826417 [GenBank] and AY826418 [GenBank] , respectively.
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Results and discussion |
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An increase in the number of ESBL-producing K. pneumoniae isolates was observed in the NICU of our university hospital during the study period (September 2002–December 2004), when 233 of a total of 554 patients (42.1%) admitted to the NICU became colonized by ESBL-producing K. pneumoniae (Figure 1). The highest values of positivity were found in rectal swabs (47.4%), followed by throat and nose cultures (35.5% and 26.4%, respectively). ESBL-producing K. pneumoniae caused 19 infections (3 sepsis, 2 pneumonias, 11 urinary tract infections, 3 ocular infections) (Figure 1). During the study period, 111 infections occurred in the ward and the four most common pathogens responsible for infections were coagulase-negative staphylococci (20.7% of infections), ESBL-producing K. pneumoniae (17.1%), Candida albicans (10.8%) and E. coli (9.9%). No pathogen was identified in 12.6% of infants diagnosed as having an infection.
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Molecular typing of all ESBL-producing K. pneumoniae isolated during the study period and of two strains isolated during the outbreak of 1996 identified three major PFGE patterns, which we designated A–C. The two isolates from the outbreak in 1996 were of type A, while the isolates obtained from September 2002 to December 2004 were predominantly of types B and C, the two types being obtained from 81 and 152 different patients, respectively. Interestingly, patients infected or colonized with strains B and C formed two consecutive temporal clusters (September 2002–December 2003 and October 2003–December 2004, respectively) linked by an overlapping period of 3 months (Figure 1). Moreover, the two epidemic clones of PFGE types B and C were isolated from throat and rectal swabs of the same patient. In all other cases, multiple isolates from surveillance swabs or clinical specimens of the same patients always showed identical PFGE patterns. The two epidemic ESBL-producing K. pneumoniae clones showed similar virulence features, PFGE clone B being responsible for 10 infections and 72 colonizations, and PFGE clone C for 9 infections and 143 colonizations.
Extensive environmental investigations were performed between November 2003 and March 2004 to identify sources and reservoirs of infection. ESBL-producing K. pneumoniae of PFGE type C was isolated from three sinks and two room surfaces in three different rooms, from two baby incubators, and from the hand of a nurse, thus suggesting horizontal transmission of the second epidemic strain from one patient to another through the hospital staff. In support of this hypothesis, it has been previously demonstrated that identical K. pneumoniae outbreak clones were isolated from infected neonates and healthcare workers' hands in NICUs.1,3 A number of control measures were introduced during the course of the outbreak, including feedback of the results of susceptibility testing and molecular typing of isolates to inform staff that there was an outbreak, enforced use of hand washing and glove use, cohorting of infected or colonized infants and their caregivers and reminding staff of the need to use appropriate antibiotic therapy in infants infected with ESBL-producing strains of K. pneumoniae. After December 2004, no new cases of ESBL-producing K. pneumoniae colonization or infection were registered in the NICU. The outbreak was considered over in January 2005 after the discharge of the last patient colonized by ESBL-producing K. pneumoniae (Figure 1).
The antimicrobial susceptibility patterns of the ESBL-producing K. pneumoniae epidemic clones of different PFGE types showed that all three were resistant to penicillins, monobactams and third-generation cephalosporins. Also, all the K. pneumoniae epidemic strains isolated from patients in the NICU were identified as ESBL producers by Etest analysis (Table 1). All three ESBL-producing K. pneumoniae epidemic clones were susceptible to second- and fourth-generation cephalosporins, piperacillin/tazobactam, carbapenems, ciprofloxacin and amikacin, but resistant to kanamycin and netilmicin. Interestingly, susceptibility to gentamicin was maintained by PFGE type A, but not by PFGE types B and C, which showed identical antibiotype. During the outbreak (September 2002–December 2004), the infants at high risk of infection in the NICU received empirical treatment based on ampicillin and gentamicin. This may have contributed to the selection of ESBL-producing K. pneumoniae epidemic clones in the NICU. Although no attempt was made in the NICU to change the empirical antibiotic treatment during the outbreak, antibiotic therapy with a carbapenem (primarily meropenem) was always adopted for ESBL-producing K. pneumoniae-infected infants. This might have favoured the successful control of the outbreak. In support of this hypothesis, a prospective multicountry study has recently demonstrated that carbapenem monotherapy is the treatment of choice for ESBL-producing K. pneumoniae infections.9
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IEF analysis of the ß-lactamase profile from K. pneumoniae epidemic strains showed two bands with pI values of 5.5 and 7.6 for PFGE type A, while identical IEF profiles, showing two bands with pI values of 5.4 and 8.3, were observed for PFGE types B and C (Table 1). PCR analysis of ß-lactamase genes showed the presence of a blaTEM gene in chromosomal DNA from all three different ESBL-producing K. pneumoniae epidemic clones and in plasmid DNA from K. pneumoniae clones of PFGE types B and C, and a blaSHV gene in plasmid DNA from all three different ESBL-producing K. pneumoniae epidemic clones. No CTX gene sequences were amplified in chromosomal or plasmid DNA of the three ESBL-producing K. pneumoniae epidemic clones. Sequence analysis of the deduced amino acid sequences identified a novel TEM enzyme in K. pneumoniae strains of PFGE type A and a TEM-1 enzyme in K. pneumoniae strains of PFGE types B and C. The novel TEM sequence was designated TEM-136 (http://www.lahey.org/studies/webt.htm) and showed the following changes from TEM-1: a serine for arginine at position 164, a threonine for alanine at position 237, a lysine for glutamic acid at position 240 and a glycine for serine at position 268. Because changes at residues 164, 237 and 240 are identical to those found in the TEM-5 enzyme, which shows ESBL activity,1 we hypothesize that TEM-136 possesses ESBL activity. Sequence analysis of the deduced amino acid sequences of the amplified blaSHV genes identified an SHV-1 enzyme in K. pneumoniae strains of PFGE type A and an SHV-12 enzyme in ESBL-producing K. pneumoniae strains of PFGE types B and C. Although TEM-136 has never been identified before, TEM-5 and TEM-10, the enzymes showing highest homology both at nucleotide and amino acid levels, have been responsible for several unrelated outbreaks of ESBL-producing organisms in the United States and since 1995 in Europe.1 Interestingly, K. pneumoniae strains isolated in our NICU during 1996 harbour TEM-136, while K. pneumoniae strains isolated during the second epidemic harbour SHV-12. In accordance with our data, the SHV-12 enzyme is highly prevalent in Italy, where it was identified in 70 of 108 (64.8%) ESBL-producing K. pneumoniae isolates during 1999.10
Conjugation experiments demonstrated successful matings for K. pneumoniae epidemic strains of PFGE types B and C at frequencies of 4 x 10–6 and 1 x 10–6 cfu/recipient cell, respectively, after selecting for resistance to ceftriaxone. The transconjugants obtained from either donor were resistant to third-generation cephalosporins, kanamycin and tobramycin, while they were susceptible to other aminoglycosides such as gentamicin and netilmicin. All transconjugants harboured a plasmid of 80 kb that showed an identical EcoRV digestion pattern and carried blaSHV-12 and blaTEM-1 genes. We were not able to obtain transconjugants in matings between K. pneumoniae strains of PFGE type A and a susceptible E. coli host when selected for ceftriaxone. Thus, we hypothesize that the outbreak described herein was sustained by the dissemination of SHV-12 ESBL through clonal expansion and horizontal gene transfer between two different K. pneumoniae clones. This is in agreement with previous data showing that dissemination of SHV-5 ESBL in a Mexican paediatric hospital was due to clonal and horizontal spread.2 Also, both SHV-5 and SHV-12 ESBL genes, which have been located in large conjugative plasmids, can be transferred in mating experiments.1,2
Finally, since ESBL-producing K. pneumoniae strains cause large and sustained colonizations and infections in the NICU environment, strict adherence to surveillance programmes is recommended to prevent colonization and infection by multidrug-resistant bacteria and antibiotic resistance dissemination.
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None to declare.
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Footnotes |
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These authors contributed equally to this work. 
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Acknowledgements |
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We thank Dr George Jacoby, Lahey Clinic, Burlington, MA, USA, for help in the identification of TEM-136 ß-lactamase, Dr Domenico Vitale from CEINGE Biotecnologie avanzate, Napoli, Italy, for technical support in DNA sequencing and Mrs Maria Grazia Catenacci for the artwork. This work was supported in part by grants from the Ministero dell'Istruzione, dell'Università e della Ricerca Scientifica e Tecnologica, Italy (PRIN 2004 to R. Z.) and the Ministero della Salute, Italy.
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References |
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