J Med Microbiol 57 (2008), 121-124; DOI: 10.1099/jmm.0.47380-0
© 2008 Society for General Microbiology
ISSN 1473-5644
Upper and lower urinary tract infection caused by Klebsiella pneumoniae serotype K2 and CTX-M-15 β-lactamase-producing serotype K1: a case report and characterization of serum killing resistance
Noriyuki Nagano1,
Christophe Cordevant2,
and
Yukiko Nagano1
1 Medical Microbiology Laboratory, Funabashi Municipal Medical Center, 1-21-1 Kanasugi, Fubanashi, Chiba 273-8588, Japan
2 Molecular Typing Center, Institut Pasteur de Lille, 1 Rue du Prof. Calmette, F-59019 Lille Cedex, France
Correspondence
Noriyuki Nagano
naganoyn{at}d3.dion.ne.jp
Received 5 May 2007
Accepted 7 September 2007
CTX-M-15 β-lactamase-producing Klebsiella pneumoniae serotype K1 was isolated from a patient with fatal upper urinary tract infection (UTI) complicated by sepsis caused by K. pneumoniae serotype K2. Transfer of a CTX-M-15 β-lactamase plasmid from the K1 to the K2 strain was observed. However, plasmid acquisition by the K2 strain did not occur in vivo, suggesting that the K1 strain might not have contributed directly to the upper UTI. In addition, effects of K serotypes and plasmid acquisition on K. pneumoniae serum resistance were examined.
Abbreviations: ESBL, extended-spectrum β-lactamase; UTI, urinary tract infection.
Present address: Food Science Division, Bio-Rad, Route de Cassel, 59114 Steenvoorde, France. 
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Case report
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A 73-year-old male patient was admitted to our medical centre with a 39 °C fever, chills, abdominal distension and ambulatory difficulties. This patient had a long history of alcoholism resulting in liver cirrhosis. On admission, the following measurements were made: leukocyte count, 35 700 µl–1 (significantly elevated); C-reactive protein level, 4.49 mg dl–1; aspartate aminotransferase, 174 IU l–1; alanine aminotransferase, 73 IU l–1; alkaline phosphatase, 881 IU l–1; blood urea nitrogen, 21 mg dl–1; and creatinine, 2.36 mg dl–1. His urine was cloudy with a pH of 5.5, and had the following characteristics: protein content, 200 mg dl–1; red blood cells, 120 per high power field; white blood cells, >200 per high power field; and bacteria, +++ per high power field. He was diagnosed with hepatic failure associated with alcoholic liver cirrhosis and urinary tract infection (UTI), while Klebsiella pneumoniae serotype K2 susceptible to broad-spectrum cephalosporins was isolated from arterial blood and urine taken upon admission and from arterial blood 34 days after admission. An ultrasonographic examination revealed hyperechoic cystic lesions in the right kidney superior border, indicative of a perinephric abscess. Biopsy cultivation revealed K. pneumoniae K2 on day 35. A dynamic computed tomography scan on day 8 showed a prostatic abscess. On day 17, multiresistant K. pneumoniae K1 was isolated from a urethral discharge culture presumably derived from the prostatic abscess. All three cultures from venous blood showed no growth, including the one collected at admission. Despite imipenem–cilastatin treatment, disseminated intravascular coagulation followed K. pneumoniae K2 sepsis and led to death on day 41.
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Microbiological methods
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Capsular K serotypes (K1–K6) were determined with Denka Seiken antisera. MICs were determined by a microdilution broth method using a WalkAway-96 SI System (NEG Combo 5 J, NEG MIC 5 J and ESBL plus panels; Dade Behring) with an inoculum of 104 c.f.u. per well. Susceptibility categories were determined according to the Clinical and Laboratory Standards Institute criteria (CLSI, 2007). The MICs for multiresistant K. pneumoniae K1 strain FM039343 are shown in Table 1
. MICs of cefotaxime and ceftazidime significantly decreased from >128 µg ml–1 to 
0.12 µg ml–1 and from 64 µg ml–1 to 0.12 µg ml–1, respectively, in the presence of 4 µg clavulanic acid ml–1, suggesting the production of class A extended-spectrum β-lactamase (ESBL) in this strain. In contrast, four K. pneumoniae K2 strains, including FM038943, derived from cultures of arterial blood taken upon admission were characterized by colonies with sticky consistency and were susceptible to broad-spectrum cephalosporins and aztreonam (Table 1). To investigate the genetic relationship among the isolates, genome typing was carried out as described previously (Nagano et al., 2003). All four K. pneumoniae K2 strains showed exactly the same XbaI-digested genomic DNA pattern, despite being isolated from different clinical sources or at different times. The restriction profile of multiresistant K. pneumoniae K1 was distinguishable from that of the four K2 strains (Fig. 1).
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Table 1. Antibiotic susceptibilities of K. pneumoniae clinical isolates and transconjugants
Values are the MIC (µg ml–1).
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Fig. 1. Agarose gel electrophoresis of XbaI-digested genomic DNA from five K. pneumoniae isolates. Restriction profiles obtained for four K. pneumoniae K2 isolates from blood (FM038943), urine, perinephric abscess aspiration and blood (left to right) were identical, suggesting a clonal lineage. These patterns were clearly different from that of the K. pneumoniae K1 strain (FM039343) isolated from urethral discharge. Lane M, lambda DNA ladder.
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Conjugal transferability of the resistance determinants was noted by using K. pneumoniae K1 as the donor, while a rifampicin-resistant (Rifr) mutant of Escherichia coli 
1037 (Iyobe et al., 1981) and an in vitro-generated Rifr K. pneumoniae K2 were the recipients, resulting in a frequency of 2.1x10–6 and 3.0x10–4 transconjugants per donor cell, respectively. Susceptibility testing revealed that the β-lactam MICs for the transconjugants were similar to those for the donor strain (Table 1
).
A unique plasmid, approximately 87 kb, was isolated from the donor and transconjugants by rapid alkaline lysis (Takahashi & Nagano, 1984). Preliminary PCR analysis (Nagano et al., 2003, 2004) on plasmid DNA isolated with the Plasmid Midi kit (Qiagen) revealed that the transconjugants showed amplification products for blaCTX-M-1 and blaTEM genes. Each of these two structural genes and flanking regions were subsequently amplified (Dutour et al., 2002; Mabilat & Goussard, 1993) and sequenced on both strands. A BLAST search (www.ncbi.nlm.nih.gov/BLAST/) revealed 100 % identity to blaCTX-M-15 (GenBank accession no. DQ302097) and blaTEM-1b (GenBank accession no. DQ058146).
To determine the contribution of each β-lactamase to antibiotic resistance, a cloning strategy using the pBC SK(+) phagemid vector (Stratagene) and E. coli XL1-Blue strain (Stratagene) was adopted. The resulting CTX-M-15 β-lactamase-producing E. coli and the parental strain shared the same profiles of resistance to β-lactams, whereas TEM-1b β-lactamase-producing E. coli was susceptible to cefotaxime and ceftazidime (data not shown).
Sensitivity of the K. pneumoniae strains to normal human serum was evaluated as described previously (Hughes et al., 1982). Bacterial suspension (500 µl) in 0.9 % NaCl (approx. 106 c.f.u. ml–1) was mixed with 1500 µl of undiluted serum (final concentration of 75 %, v/v) with or without 30 µg cefotaxime ml–1 corresponding maximum plasma concentration after 1 g intravenous infusion. Viable counts were determined at the beginning and after 1, 2 and 3 h of 37 °C incubation. The assay was performed in triplicate for each strain, and the results showed intermediate resistance of K. pneumoniae K2 (Fig. 2b), whereas K. pneumoniae K1 was rapidly killed within 2 h of incubation with human serum (Fig. 2a). It is noteworthy that among two K. pneumoniae K2 strains, an ESBL producer showed a higher level of serum resistance by growing equally well in normal and heat-inactivated serum. The ESBL-producing K2 strain lost its serum resistance in combination with cefotaxime, even at subinhibitory concentrations (Fig. 2b, c).
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Discussion
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Bacterial infection of the prostate gland might have occurred from an ascending urethral infection or by reflux of infected urine into prostatic ducts emptying into the posterior urethra. Invasion of rectal bacteria by direct extension or by lymphogenous or haematogenous spread may also constitute other possible routes (Domingue & Hellstrom, 1998). Since the ESBL producer isolated from a purulent urethral discharge was not detected in other clinical samples including blood, urine, perinephric abscess and sputum, the prostatic abscess is the most probable source of this strain, while infected urine is the less likely source. In vivo transfers of ESBL-encoding plasmids have been previously reported (Neuwirth et al., 2001). The mating assay showed that the plasmid transfer frequency from the K. pneumoniae K1 strain to the K2 strain was constant, about a hundred times reproducibly higher than transfer from the K1 strain to E. coli 1037. However, the acquisition of the ESBL-encoding plasmid by the K2 strain did not occur in vivo, which also suggests that the K1 strain might not have contributed directly to the upper UTI in this case.
ESBL-producing K. pneumoniae has been increasingly reported worldwide. However, few studies have analysed the association between virulence and ESBL production in K. pneumoniae (Di Martino et al., 1997; Sahly et al., 2004). Mizuta et al. (1983) have previously shown that K. pneumoniae strains expressing capsular serotypes K1 and K2 are particularly virulent in mice. Moreover, it is well established that the K2 serotype is among the most common capsular serotypes detected from patients with UTI, pneumonia or bacteraemia (Podschun & Ullmann, 1998). In this report, the DNA restriction profiles were identical among all four K. pneumoniae K2 strains, suggesting a persistent infection of the upper urinary tract, despite their in vitro sensitivity to therapeutic agents. To assess an association of K serotypes and ESBL production with pathogenicity, serum sensitivity was taken as a measure of virulence for K. pneumoniae. The results indicate that serum killing resistance was significantly higher in K. pneumoniae K2 than in K1. In particular, acquisition of the ESBL-encoding plasmid enhanced K2 serum resistance. It is uncertain whether serum resistance in K2 ties in with clinical course in this case. However, the possible acquisition of ESBL-encoding plasmids among more virulent K. pneumoniae serotypes might result in severe therapeutic problems in immunocompromised hosts, while persistent infection may substantially increase the risk of progression to nosocomial spread.
This case is remarkable in that K. pneumoniae strains with different capsular types, susceptibility profiles and serum-mediated killing resistance were isolated from the same patient with severe UTI.
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Case report
Microbiological methods
), NHS containing 30 µg cefotaxime ml–1 (
) or heat-inactivated NHS (
). Results shown are the mean from three independent experiments.