Metallo-β-lactamases (MBLs) are the enzymes capable of hydrolyzing almost the full range of β-lactam-antibiotics – penicillins, cephalosporins and carbapanems. Some bacterial strains, e.g. Stenotrophomonas maltophilia, possess natural MBLs; yet the real threat is the occurrence and spread of these enzymes amongst serious human pathogens, which is favoured by the presence of genes encoding them on conjugative plasmids. The first strain producing the acquired MBL, i.e. Pseudomonas aeruginosa, was isolated in Japan in 1988 [1]. Since that time, the second half of 1990, in particular, the presence and spread of MBL-positive strains have been observed in many countries [2, 3, 4]. At present, 8 types of acquired MBLs are distinguished: IMP, VIM, SPM, GIM, SIM, KHM, AIM and DIM. Their production is most commonly associated with the Enterobacteriaceae family [2, 3, 5, 6], although they still predominate amongst the non-fermenting rods.

The risk of hospital infection in ITUs is 4-5 times higher than in other wards [7]. Infections in ITU patients are characterized by severe course and high mortality [8]. An important factor increasing the bacterial resistance to antimicrobial drugs in ITUs is the selection of resistant strains due to frequent use of broad-spectrum antibiotics [9].

The present report describes the isolation of metallo-β-lactamase producing Klebsiella pneumoniae in a patient requiring intensive therapy.

CASE REPORT

Four strains of Klebsiella pneumoniae isolated in a 61-yr-old Caucasian patient hospitalized in the Department of Anaesthesiology and Intensive Therapy, Teaching Hospital in Bydgoszcz were studied. Each strain was isolated from different clinical materials: urine, wound smear, blood and abdominal fluid. The patient was admitted to hospital on an emergency basis due to acute lower limb ischaemia and was qualified for urgent vascular surgery. The history revealed arterial hypertension and ischaemic heart disease. Before the implantation of an aortobifemoral prosthesis, a single prophylactic dose of cefuroxime was administered.  Immediately after surgery, the patient was admitted to ITU due to acute respiratory failure. During the first ITU day, directly after blood sampling, bronchoalveolar lavage (BAL) and urine collection, the patient received piperacillin with tazobactam. On day 2, the renal replacement therapy (CVVH/HVCVVH/HDF) was initiated due to acute renal failure in the course of rhabdomyolysis, which was continued throughout the ITU stay. Empiric antibiotic therapy was carried out until day 6; microbiological results were negative. However, due to the patient`s worsening condition, increasingly high indices of inflammatory reaction and generalized infection, meropenem and vancomycin were included. The treatment was continued for 7 days. On day 9, septic shock was diagnosed based on clinical symptoms without microbiological confirmation. 

Since the pathogen was undetected and vital parameters did not normalize, on day 15 cefepime was included and recombined activated protein C administered. On day 17, the first positive BAL was obtained, from which Pseudomonas aeruginosa resistant to carbapenems was isolated. On day 19, due to the patient`s severe condition, it was decided to widen the empiric antibiotic therapy against Gram-positive bacteria and fungi. Linezolid, caspofungin and amphotericin B were included. On day 23, another positive culture was obtained. Pseudomonas aeruginosa susceptible to ciprofloxacin was isolated. Cefepime was discontinued and ciprofloxacin introduced with the earlier treatment continued. On day 24, positive culture of abdominal purulent contents was obtained. Stenotrophomonas maltophilia sensitive to cephalosporins, aminoglycosides and cotrimoxazole was isolated. On day 26, gastrointestinal haemorrhage developed and the patient was qualified for surgery. On day 28, vancomycin, discontinued on day 13, was reintroduced.

On day 29, the positive urine culture was obtained with Klebsiella pneumoniae susceptible only to imipenem and aztreonam isolated. Imipenem was included; during the further 2 days, another bleeding from the gastrointestinal tract occurred. During the third procedure, the patient underwent total gastrectomy. On treatment day 30, positive blood, urine, abdominal fluid, BAL and wound smear cultures were obtained. Klebsiella pneumoniae susceptible only to aztreonam and gentamicin and Acinetobacter baumannii susceptible to imipenem were isolated from blood. Acinetobacter baumannii sensitive only to imipenem was also isolated from BAL. From the remaining isolates, Klebsiella pneumoniae susceptible to aztreonam, gentamicin and tetracycline was identified.  Strains isolated from blood, urine and wound smears were intermediate-susceptible to amikacin.

On day 30, the patient died with the symptoms of multi organ failure in the course of septic shock.

The strains were identified based on the results of biochemical reactions included in ID32E tests (bioMérieux). The drug-susceptibility of strains was determined using the disc-diffusion method and E-tests (AB Biodisk) [10]. The values of minimal inhibitory concentrations (MICs) of antibiotics the strains were susceptible or medium-susceptible to, are presented in Table 1. All Klebsiella pneumoniae isolates were resistant to imipenem and meropenem and their MIC values were > 32 μg mL-1. The capacity to produce MBL was determined using the double disc test [10]. For a single MBL-positive isolate from blood, PCR hyplex MBL ID (BAG Health Care) was used and MBL from the VIM family (blaVIM) identified. The conjugative test involving an attempt to transfer the MBL encoding gene (blaVIM ) to the recipient Escherichia coli A15 strain was performed as described earlier [6] using meropenem 2 μg mL-1 in the selective medium. The test was positive, which meant that blaVIM was located in the plasmid capable of conjugative transfer.

DISCUSSION

Pseudomonas aeruginosa strains with acquired MBLs have occurred in Poland since 1998-2000 [6, 11, 12]. As far as we know, Klebsiella pneumoniae isolates described in the present report were the first such microorganisms producing MBLs in our country. Such strains have already been described in Europe, Africa, Asia and both Americas [13, 14]. In some countries, especially in Greece, their incidence in Klebsiella pneumoniae populations in ITUs increased from 1% to 50% in the years 2001-2006 [15], which was mainly associated with uncontrolled spread of a single pathogen clone with VIM-1 (VPKP) [16]. This evidences a serious epidemiological threat of MBL-positive Enterobacteriaceae rods when strategies of hospital infection control are faulty. According to the recommendations of the National Consultant for microbiological diagnostics, the detection of this mechanism of resistance should be included in the routine diagnostic procedures and each strain suspected of producing MBLs should be sent to the National Referential Centre for Drug Susceptibility of Microorganisms for confirmation with reference methods [10]. The majority of MBL-producing strains have a similar worldwide phenotype of drug susceptibility. The strains discussed in our report were susceptible to aztreonam, gentamicin and tetracycline. There are no clinical trials available confirming the efficacy of these antibiotics in the treatment of infections with MBL-positive strains.

It should be remembered that carbapanems remain one of very few therapeutic options in severe infections with multi-resistant strains, especially in ITU patients. However, their use becomes markedly limited due to the occurrence of MBL-positive strains.

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REFERENCES

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6.    Gniadkowski M, Schneider I, Jungwirth R, Hryniewicz W, Bauernfeind A: Ceftazidime-resistant Enterobacteriaceae isolates from three Polish hospitals: identification of three novel TEM- and SHV-5-type extended-spectrum b-lactamases. Antimicrob Agents Chemother 1998; 42: 514-520.

7.    Singh AK, Sen MR, Anupurba S, Bhattacharya P: Antibiotic sensitivity pattern of the bacteria isolated from nosocomial infections in ICU. J Commun Dis 2002; 34: 257-263.

8.    Rudnicka J, Wróblewska M, Marchel H, Łuczak M: Częstość występowania i lekooporność pałeczek z rodziny Enterobacteriaceae izolowanych od pacjentów hospitalizowanych na oddziałach intensywnej terapii. Med Dośw Mikrobiol 2005; 57: 185-191.

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11.    Patzer J, Toleman MA, Deshpande LM, Kamińska W, Dzierżanowska D, Bennett PM, Jones RN, Walsh TR: Pseudomonas aeruginosa strains harbouring an unusual blaVIM-4 gene cassette isolated from hospitalized children in Poland (1998-2001). J Antimicrob Chemother 2004; 53: 451-456.

12.    Fiett J, Baraniak A, Mrówka A, Fleischer M, Drulis-Kawa Z, Naumiuk Ł, Samet A, Hryniewicz W, Gniadkowski M: Molecular epidemiology of the acquired metallo-b-lactamase-producing bacteria in Poland. Antimicrob Agents Chemother 2006; 50: 880-886.

13.    Walsh TR, Toleman MA, Hryniewicz W, Bennett PM, Jones RN: Evolution of an integron carrying blaVIM-2 in Eastern Europe: report from the SENTRY Antimicrobial Surveillance Program. J Antimicrob Chemother 2003; 52: 116-119.

14.    Luzzaro F, Docquier JD, Colinon C, Endimiani A, Lombardi G, Amicosante G, Rossolini GM, Toniolo A: Emergence in Klebsiella pneumoniae and Enterobacter cloacae clinical isolates of the VIM-4 metallo-beta-lactamase encoded by a conjugative plasmid. Antimicrob Agents Chemother 2004; 48: 648-650.

15.    Vatopoulos A: High rates of metallo-beta-lactamase-producing Klebsiella pneumoniae in Greece – a review of the current evidence. Eurosurveillance 2008; 13: 1-6.

16.    Loli A, Tzouvelekis LS, Tzelepi E, Carattoli A, Vatopoulos AC, Tassios PT, Miriagou V: Sources of diversity of carbapenem resistance levels in Klebsiella pneumoniae carrying blaVIM-1. J Antimicrob Chemother 2006; 58: 669-672.

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Address:

*Alicja Sękowska
Katedra i Zakład Mikrobiologii,
Collegium Medicum w Bydgoszczy
Uniwersytetu Mikołaja Kopernika w Toruniu
ul. M. Skłodowskiej-Curie 9, 85-094 Bydgoszcz
tel.: 0-52 585-44-80

Received: 05.09.2009
Accepted: 04.12.2009