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Keywords:

  • Klebsiella pneumoniae ST512;
  • blaKPC-3;
  • multidrug resistance;
  • outbreak;
  • blaTEM

Abstract

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results and discussion
  5. Funding
  6. Conflict of interest
  7. References

Strains of Klebsiella pneumoniae producing KPC-carbapenemase have emerged as one of the most important multidrug-resistant Gram-negative nosocomial pathogens. Here, we report the first isolation and subsequent dissemination of a K. pneumoniae ST512 producing KPC-3 carbapenemase in a hospital in southern Italy. Isolates were obtained from blood, throat swabs, sputum, catheters, and urine of patients admitted to different hospital wards. Antimicrobial MICs were determined for all isolates by automated systems and confirmed by Etest. Carbapenemase production was confirmed by the modified Hodge test and by a disc synergy test, and carbapenemase genes were investigated by PCR. All isolates were characterized by pulse-field gel electrophoresis (PFGE) and multilocus sequence typing (MLST) analysis. Most isolates were multidrug resistant with exception of some isolates intermediately susceptible to gentamicin, tigecycline, and trimethoprim-sulfamethoxazole. PCR analysis showed that isolates harbored the blaKPC-3 gene associated with blaTEM and blaSVH. PFGE and MLST showed that all isolates belonged to the same ST512 clone recently described in Israel.

In the last few years, Klebsiella pneumoniae strains producing KPC (K. pneumoniae carbapenemase) enzymes have emerged as one of the most important multidrug-resistant (MDR) Gram-negative pathogens in hospitalized patients [1]. The first isolation of KPC-producing strains was reported in the USA [2] and since then, it has been reported in other countries including Italy [3-5]. KPC beta-lactamases, belonging to the Amber class A, include ten variants, of which KPC-3 and KPC-2 are predominant. The most frequently isolated sequence type (ST) of K. pneumoniae associated with KPC-2 or KPC-3 enzyme production is ST258, with a global distribution [6] and most specifically in Italy [3-5]. Recently, Warburg and coworkers described the isolation of a K. pneumoniae ST512 carrying the KPC-3 enzyme in Israel [7]. Here, we report an outbreak of a carbapenem-resistant K. pneumoniae strain in a hospital in southern Italy, occurring between February 2011 and January 2012.

Materials and methods

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results and discussion
  5. Funding
  6. Conflict of interest
  7. References

Clinical isolates

The 43 K. pneumoniae isolates included in the study were detected from February 2011 to January 2012 at Policlinico Federico II, University of Naples, Italy. The identifications were performed by automatic VitekII system (bioMerieux, Marcy L'Etoile, France) and confirmed by a MALDI-TOF spectrometer (Bruker Daltonics, Bremen, Germany). The antimicrobial susceptibility testing of all isolates was determined using VitekII system and results were interpreted according to EUCAST guidelines [8]. Confirmation of carbapenemase production was performed by the modified Hodge test and by carbapenemase inhibition tests (Rosco Diagnostica, Taastrup, Denmark).

PCR and sequencing of β-lactamase genes

Specific primers were used to amplify and sequence the blaKPC and other resistance genes, according to Dallenne et al. [9]. PCR reactions were performed directly on 2–3 colonies picked from a pure culture using 10 pmol of each primer, 200 μM dNTP, 3 mM MgCl2, and 0.5 U Taq polymerase. The amplification program consisted of the following phases: initial denaturation at 94 °C for 10 min; 30 cycles of denaturation at 94 °C for 2 min, annealing at 57 °C for 1 min, and elongation at 72 °C for 2 min; final elongation at 72 °C for 10 min. Subsequently, the amplification products were sequenced at the Center for Genetic Engineering (CEINGE) of Naples using the same PCR primers. Gene resistance sequences were determined by alignment with NCBI database using the BLAST program; alleles and Multilocus sequence typing (MLST) profiles were determined by comparison with the K. pneumoniae database at www.pasteur.fr/mlst (http://www.pasteur.fr/recherche/genopole/PF8/mlst/Kpneumoniae.html).

Clonal analysis by PFGE

Isolates were grown overnight and suspended in SE buffer (75 mM NaCl, 25 mM EDTA, pH 7.5). The cell suspensions (4 McFarland units) were mixed with an equal volume of 2% low-melting point agarose, molded into plugs at 4 °C, and lysed with lysis buffer (1% N-lauryl sarcosine, 0.5 M EDTA, pH 8) to which Proteinase K (500 μg/mL) had been added. The DNA contained into each mold was digested by 20 U of XbaI restriction enzyme (New England Biolabs, Beverly, MA, USA), in accordance with the manufacturer's instructions. Macrorestriction fragments were separated using the CHEF-DR III pulse-field gel electrophoresis (PFGE) system (Bio-Rad, Hercules, CA, USA) at 10 °C for 20 h, with an initial switch time of 5 s and a final switch time of 35 s, at a field strength of 6 V/cm. A ladder of lambda phage DNA concatemers was used as a size marker. Restriction profiles were analyzed according to Tenover's criteria [10].

Results and discussion

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results and discussion
  5. Funding
  6. Conflict of interest
  7. References

The first isolate producing carbapenemase was obtained from sputum of a patient with cystic fibrosis and admitted to the Pediatric department in February 2011; for 10 months after the first isolation, there was a clonal dissemination which affected other 42 patients admitted to different wards (ICUs, Surgery, Hematology, Pediatrics, Cardiac Surgery, Neurology, Nephrology, and the Medical Division).

Klebsiella pneumoniae strains caused bloodstream infections in 20/43 patients, respiratory infections in 28/43 patients (10 of which also had a bloodstream infection), urinary infection in three patients and surgical site infection in another; the other patients admitted in the same wards during the period of outbreak were screened for colonization by rectal swabs, and other 19 resulted positive for the colonization with K. pneumoniae, but they did not develop any infection. Colonization of the rectal or urinary tract by K. pneumoniae was found to persist in seven patients even after recovery from bloodstream or respiratory infections (Table 1). It was not possible to establish whether patients who developed serious infections in the early phase of the outbreak were already colonized at the time of admission. All patients were treated with antibiotic therapy; most of them were discharged from the hospital, although some were still colonized at discharge. A termination of the outbreak was declared in March 2012, but continued surveillance showed that carbapenem-resistant K. pneumoniae strains tended to remain in the hospital environment and many patients were found to be colonized at admission to hematologic or pediatric units.

Table 1. Characteristics of all blaKPC-3-producing Klebsiella pneumoniae isolates
IsolateMedical wardSpecimensPatient diagnosisAntibiotic resistance MIC (mg/L)
CAZCTXFEPIMPMEMETPLVXGMTZPTGCSTX
  1. Medical wards: ICU, intensive care unit; NICU, neonatal intensive care unit; Pediatric FC, pediatric ward for cystic fibrosis patients.

  2. Patient diagnosis: RI, respiratory infection; SSI, surgical site infection; BI, bloodstream infection; UI, urinary infection.

  3. Specimens: S, sputum; BA, bronchial aspirate; B, blood; U, urine; AW, abdominal wound.

  4. Antibiotics: CAZ, ceftazidime; CTX, cefotaxime; FEP, cefepime; IMP, imipenem; MEM, meropenem; ETP, ertapenem; LVX, levofloxacin; GM, gentamicin; TZP, piperacillin-tazobactam; TGC, tigecycline; STX, trimethoprim-sulfamethoxazole.

  5. MIC breakpoints according to EUCAST (S≤/R): CAZ 1/4, CTX 1/2, FEP 1/4, IMP 2/8, MEM 2/8, ETP 0.5/1, LVX 1/2, GM 2/4, TZP 8/16, TGC 1/2, STX 2/4.

KP1Pediatric FCSRI323232>8>8>188>64/412/38
KP2SurgeryAWSSI646464>8>8>188>64/414/76
KP3NephrologyUUI646464>8>8>184>64/414/76
KP4NICUBBI323232>8>8>144>64/42>4/76
KP5ICUB, BABI, RI646464>8>8>148>64/412/38
KP6PediatricsBARI323232>8>8>188>64/412/38
KP7SurgeryUUI323232>8>8>188>64/412/38
KP8ICUBARI646464>8>8>184>64/414/76
KP9Cardiac SurgeryBBI323232>8>8>144>64/42>4/76
KP10ICUBA, BBI, RI323232>8>8>144>64/41>4/76
KP11ICUBA, BBI, RI323232>8>8>144>64/422/38
KP12ICUBBI323232>8>8>144>64/41>4/76
KP13Pediatric FCSRI323232>8>8>188>64/412/38
KP14Cardiac SurgeryBARI646464>8>8>184>64/42>4/76
KP15NeurologyBA, URI646464>8>8>148>64/412/38
KP16Cardiac SurgeryBARI323232>8>8>144>64/42>4/76
KP17ICUBARI646464>8>8>148>64/412/38
KP18HematologyBBI323232>8>8>144>64/42>4/76
KP19PediatricsBARI646464>8>8>184>64/42>4/76
KP20HematologyBBI646464>8>8>184>64/42>4/76
KP21ICUBBI646464>8>8>184>64/42>4/76
KP22HematologyBBI, RI646464>8>8>184>64/42>4/76
KP23ICUBA, B, UBI, RI646464>8>8>184>64/42>4/76
KP24ICUBABI323232>8>8>144>64/422/38
KP25ICUBA, B, UBI, RI323232>8>8>144>64/42>4/76
KP26HematologyBBI646464>8>8>184>64/42>4/76
KP27Medical DivisionUUI323232>8>8>144>64/422/38
KP28Cardiac SurgeryBBI646464>8>8>148>64/412/38
KP29NICUBARI646464>8>8>184>64/42>4/76
KP30Cardiac SurgeryBARI646464>8>8>184>64/42>4/76
KP31ICUBA, URI646464>8>8>184>64/42>4/76
KP32ICUBBI646464>8>8>184>64/42>4/76
KP33HematologyBBI323232>8>8>144>64/41>4/76
KP34ICUBA, BBI, RI646464>8>8>184>64/42>4/76
KP35ICUBA, BBI, RI646464>8>8>184>64/42>4/76
KP36ICUBA, BBI, RI646464>8>8>184>64/42>4/76
KP37ICUBARI646464>8>8>184>64/42>4/76
KP38ICUBARI646464>8>8>184>64/42>4/76
KP39PediatricsSRI646464>8>8>184>64/42>4/76
KP40ICUBA, URI323232>8>8>144>64/422/38
KP41Pediatric FCSRI646464>8>8>184>64/42>4/76
KP42Pediatric FCSRI323232>8>8>144>64/42>4/76
KP43ICUBA, BBI, RI646464>8>8>148>64/412/38

The isolates were resistant to piperacillin/tazobactam, all tested cephalosporins and carbapenems at a high level (IMP > 8, MEM > 8, ETP > 1); most of the isolates were intermediately susceptible to tigecycline (MIC 2 mg/L), gentamicin (MIC 4 mg/L), and trimethoprim-sulfamethoxazole (MIC 4/76 mg/L; Table 1); all isolates were susceptible to colistin (MIC < 0.5 mg/L). Isolates from the urinary tract were also tested for antibiotics used for non-complicated urinary infections and were resistant to nitrofurantoin (MIC > 64 mg/L) and susceptible to fosfomycin (MIC < 16 mg/L).

A modified Hodge test and carbapenemase inhibition tests were performed to discriminate the Amber classes of carbapenemases. Isolates were positive in the modified Hodge test and in the boronic acid inhibition test which is indicative of a KPC beta-lactamase (blaKPC, Amber class A). Analysis of blaKPC genes by PCR and sequencing revealed the presence of the blaKPC-3 gene in all isolates. The isolates were also screened for the presence of other ESBL genes showing the additional presence of the blaTEM and blaSVH genes, while the plasmid-mediated AmpC beta-lactamase genes (AAC,FOX, MOX, DHA) were absent. All isolates were genotyped by PFGE after XbaI digestion and were indistinguishable (Fig. 1). MLST was performed according to the K. pneumoniae MLST website revealing that all isolates belonged to the ST512 (allelic profile: 54-3-1-1-1-1-79), that is a single locus variant of the ST258 (allelic profile: 3-3-1-1-1-1-79) clone associated with the dissemination of KPC enzymes worldwide.

image

Figure 1. PFGE patterns showing the genetic relatedness of 10 blaKPC3-producing Klebsiella pneumoniae isolates from 10 different wards of hospital (lane 1–10). M: Molecular weight marker 50–1000 kb from λ phage (Sigma Aldrich, St. Louis, MO, USA).

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In this study, we have reported for the first time the isolation and dissemination of a K. pneumoniae ST512 strain in a hospital in southern Italy. The ST512 strain was isolated in Israel in late 2005, where it caused a nationwide outbreak [7]. Strain ST512 showed a single mutation with respect to the more widespread ST258, i.e. the C176A transversion in the gapA locus. The strain isolated in Israel carried a pKpQIL plasmid encoding KPC-3 and TEM-1 previously isolated in a ST258 strain [11], a second plasmid encoding aac(6′)-Ib [12] and a blaSVH gene in the chromosome. The strain isolated in our hospital showed the same beta-lactamase genes and the identical pattern of resistance. In addition, our isolates were found to be sensitive to tigecycline and colistin, which was not reported for the Israeli clone. ST512 is uncommon among KPC-producing strains isolated in Europe [13-15], but it was widely described in Israel. It has not been possible to correlate the first case of ST512 infection in our hospital with a history of a recent journey to Israel or countries where this clone could be widespread. However, this report sounds an alarm on the control of the worldwide and often epidemic spread of carbapenemase-producing enterobacteriaceae and points out the need for accurate surveillance measures against a further dissemination of this clone from Italy to other European regions.

Conflict of interest

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results and discussion
  5. Funding
  6. Conflict of interest
  7. References

The authors who have taken part in this study declared that they do not have anything to disclose regarding funding or conflict of interest with respect to this manuscript.

References

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results and discussion
  5. Funding
  6. Conflict of interest
  7. References
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