Abstract

Klebsiella pneumoniae has adapted to the extensive and intensive use of antibacterial drugs in hospitals. Over the last 30 years, K. pneumoniae went from having partial resistance to ampicillin and narrow-spectrum cephalosporins to current pandemic resistance to broad-spectrum cephalosporins due to extended-spectrum beta-lactamase production as well as multidrug resistance to penicillins, cephalosporins, and monobactams⁽¹⁾1 Braykov B, Eber MR, Klein EY, Morgan DJ, Laxminarayan R. Trend resistance to carbapenens and third generation cephalosporins among clinical isolates of K. pneumoniae in the United States, 1999-2010. Infect Control Hosp Epidemiol 2013; 34:259-268.. Likewise, carbapenemase-producing strains such as Klebsiella pneumoniaecarbapenemase (KPC) and New Delhi metallo-beta-lactamase producers, which are not susceptible to imipenem or other carbapenem drugs, have rapidly emerged and spread worldwide⁽²⁾2 Cabral AB, Melo RC, Maciel MA, Lopes AC. Multidrug resistance genes, including bla (KPC) and bla (CTX-M-2), among Klebsiella pneumoniae isolated in Recife, Brazil. Rev Soc Bras Med Trop. 2012; 45:572-578.⁽³⁾3 Rapp RP, Urban C. K. pneumoniae carbapenemases in Enterobacteriaceae: history, evolution, and microbiology concerns. Pharmacotherapy 2012; 32:399-407.. Colistin and polymyxin B are among the few remaining drugs able to combat these multidrug-resistant strains and having satisfactory efficacy in the treatment of patients infected with KPC-producing K. pneumoniae⁽⁴⁾4 Falagas ME, Rafailids PI, Ioannidou E, Aleixou VG, Mattraiou DK, Karageorpoulos DE, et al. Colistin therapy for microbiologically documented multidrug-resistant Gram-negative bacterial infections: a restrospective cohor study of 258 patients. Int J Antimicrob Agents 2010; 35:194-199.. However, KPC producers also resistant to polymyxins have recently been detected⁽⁵⁾5 Marchain D, Chopra T, Poque RM, Perez F, Huyer AM, Rudin S, et al. Outbreak of colistin-resistant, carbapenem-resistant, K. pneumoniae in metropolitan Detroit, Michingan. Antimicrob Ag Chemother 2011; 55:593-599..

Here, we report the cases of four patients involved in a cluster of polymyxin-resistant and KPC-producing K. pneumoniae in order to clarify the factors associated with the induction and dissemination of this extensively drug- resistant strain.

Infection by polymyxin-resistant KPC-producing K. pneumoniae occurred in patients admitted to the University Hospital, Faculty of Medicine of Ribeirão Preto, University of São Paulo, Brazil - a public university hospital that provides tertiary medical care - in November 2011. The hospital units where transmission and/or isolation of the extensively drug-resistant clone occurred are located on the 6^th floor (hematology ward and bone marrow transplant unit), 5^th floor (geriatric ward), and 2^nd floor (intensive care unit [ICU]). Clinical and epidemiological data were obtained retrospectively from the patient's medical records.

Bacterial identification and initial susceptibility testing were performed by using the VITEK 2 automated microbial identification system (BioMérieux, Mercy L'Etoile, France). E-test^(r) strips (BioMérieux) were used to determine the minimum inhibitory concentrations (MICs) of colistin and polymyxin B for KPC-producing K. pneumoniae isolates in duplicate. The MICs for colistin were interpreted according to the European Committee on Antimicrobial Susceptibility Testing (EUCAST) guidelines⁽⁶⁾6 The European Committee on Antimicrobial Susceptibility Testing (EUCAST). Breakpoints tables for interpretation of MICs and zone diameters. Version 3.1. EUCAST; 2013. (Cited 2015 February). Available at http://www.eucast.org.
http://www.eucast.org... . The breakpoints proposed for Acinetobacter baumannii ⁽⁷⁾7 Lat A, Clock SA, Wu F, Whittier S, Della-Latta P, Fauntleroy K, et al. Comparison of polimyxin B, tigecycline, cefepime and meropenem MICs for KPC-producing K. pneumoniae by broth microdilution, Vitek 2, and Etest. J Clin Microbiol 2011; 49:1795-1798. were adopted for the MICs of polymyxin B.

Polymyxin-resistant and KPC-producing K. pneumoniae were isolated from the four patients from December 8, 2011 to January 27, 2012. All patients had serious underlying diseases, and three were receiving immunosuppressive drugs ( Table 1 ). All four patients had been hospitalized previously. Two were readmitted to the bone marrow transplant unit, one to the hematology ward, and one to the geriatric ward; the latter two were also hospitalized for some time in the ICU ( Figure 1 ). Their clinical data are presented below.

Figure 1:
A cluster of carbapenem- and polymyxin-resistant Klebsiella pneumoniae cases in four patients: hospital ward, transference to intensive care unit, and time of isolation of carbapenemase- producing K. pneumoniae susceptible (x) or resistant to polymyxins (X), and death (+).

Case 1

Case 1 was of a 39-year-old woman with acute myeloid leukemia who was admitted to the hematology ward for chemotherapy. She developed pulmonary aspergillosis and bloodstream infection with KPC-producing K. pneumoniae as a consequence of neutropenia and had been treated with voriconazole, polymyxin B (25 days), and tigecycline (14 days). Although KPC-producing K. pneumoniae was further isolated within 10 days of the initiation of treatment with polymyxin B, both infections were controlled. The patient was readmitted and received to bone marrow transplantation. She developed another bloodstream infection with KPC-producing K. pneumoniae and was treated again with polymyxin B (25 days) plus tigecycline (21 days). The two polymyxin B treatments were separated by 50 days. The patient developed severe mucositis after bone marrow transplantation, and polymyxin-resistant and KPC-producing K. pneumoniae was isolated from an oropharyngeal ulcer 14 days after restarting polymyxin B. However, she died from a new bloodstream infection with multi-susceptible K. pneumoniae. Throughout both admissions, polymyxin-susceptible and KPC-producing K. pneumoniae was isolated from 10 rectal swabs, revealing persistent colonization for more than 100 days.

Case 2

Case 2 was of a 74-year-old woman who had been suffering from Sheehan syndrome for 30 years and developed acute myeloid leukemia. While receiving chemotherapy, she suffered several complications and consequently stayed in the ICU for 20 days. She developed febrile neutropenia and received courses of antibacterial drugs including polymyxin B. On the 42^nd day of hospitalization, a rectal swab culture revealed KPC-producing K. pneumoniae. Polymyxin-resistant and KPC-producing K. pneumoniae was subsequently isolated from two blood cultures on the 49^th day and a urine culture on the 53^rd day. The patient was being treated with meropenem, followed by the addition of amikacin. However, she developed septic shock and died.

Case 3

Case 3 was of a 36-year-old man who experienced acute myeloid leukemia relapse after bone marrow transplantation and was readmitted to the bone marrow transplantation ward for chemotherapy. He developed febrile neutropenia, tonsillitis, pulmonary infiltrate, and cellulitis at the venous catheter implantation site. Antibacterial agents and voriconazole were administered continuously. On the 49^th day of hospitalization, polymyxin-resistant and KPC-producing K. pneumoniae was isolated from two consecutive blood cultures. The patient was treated empirically with polymyxin B and tigecycline for two days, but suffered septic shock and died.

Case 4

Case 4 was of a 64-year old woman who had chronic arterial disease, diabetes mellitus, and chronic renal failure. She was admitted to the geriatric ward and subsequently transferred to the ICU seven days after case 2 left the unit. She underwent surgery for revascularization of the right leg. However, a surgical site infection occurred, from which A. baumannii and KPC-producing K. pneumoniae were isolated. Empirical antibiotics therapy was replaced with polymyxin B and gentamicin. After 19 days of polymyxin B treatment, she developed fever and cellulitis at the venous catheter insertion site. The catheter was removed, and polymyxin-resistant and KPC-producing K. pneumoniae was isolated from its tip. The patient was treated with tigecycline, and her fever stopped.

Polymyxin B 20,000 IU/Kg/day was administered for cases 1, 2, and 4. The microbiological data of the patients are shown in Table 2. In addition to resistance to polymyxin B and colistin, three KPC-producing K. pneumoniae isolates were also resistant or had intermediate susceptibility to tigecycline. However, all isolates were susceptible to amikacin.

The present report describes the clinical and hospital factors associated with the emergence and transmission of polymyxin-resistant K. pneumoniae. Three previously studied isolates (cases 2-4) carried the bla _KPC2 and gnrS1 genes and belong to sequence type (ST)-11, an internationally occurring high-risk clone⁽⁸⁾8 Andrade LN, Vitali L, Gaspar GG, Bellíssimo-Rodrigues F, Martinez R, Darini AL. Expansion and evolution of a virulent, extensively drug- resistant (polymyxin B- resistant), QnrS1-, CTX-M-2-, and KPC-2-producing Klebsiella pneumoniae ST11 international high- risk clone. J Clin Microbiol2014; 52:2530-2535.; these bacterial isolates simultaneously carry genes encoding virulent phenotypes and related to multidrug resistance. Different multidrug-resistant K. pneumoniae clones associated with patient colonization or infection, such as extended-spectrum and CTX-M beta-lactamase producers have been detected in the hospital where the cluster occurred⁽⁹⁾9 Minarini LAR, Clímaco EC, Guimarães DB, Ferreira JC, Palazzo ICV, Martinez R, et al. Clonal transmission of ESBL-producing Klebsiella spp at a university hospital in Brazil. Curr Microbiol 2008; 56:587-591.. The isolation of carbapenem-resistant K. pneumoniae from blood cultures and other samples has increased in recent years; these isolates were characterized as KPC-2 producers as well as ST-258, ST-11, and ST-48 clones⁽¹⁰⁾10 Andrade LN, Curiao T, Ferreira JC, Longo J, Clímaco EC, Martinez R, et al. Dissemination of bla KPC-2 by the spread of K. pneumoniae clonal complex 258 clones plasmids (Inc F II, Inc L/M) among Entrerobacteriaceae species in Brazil. Antimicrob Ag Chemother2011; 55:3579-3583.. This epidemiological change is similar to those that have occurred in hospitals in other regions and countries, i.e., increases in cases of infection attributed to the ST-11 and other clones of KPC-producing K. pneumoniae ⁽¹¹⁾11 Pereira PS, Araujo CF, Seki LM, Zahuer V, Carvalho-Assef AP, Asensi MD. Update of the molecular epidemiology of KPC-2-producing K. pneumoniae in Brazil: spread of clonal complex 11 (ST 11, ST 437 and ST 340). J Antimicrob Chemother 2013; 68:312-316..

The dissemination of Gram-negative bacilli resistant to imipenem and other carbapenem drugs led to increased use of polymyxins for infected patients in hospitals. However, previous use of colistin is the only independent factor for the isolation of Gram-negative bacilli resistant to this antibiotic⁽¹²⁾12 Matthaiou DK, Michalopoulos A, Rafailidis PI, Karageorgopoulos DE, Papaionnou V, Nemi G, et al. Risk factors associated with the isolation of colistin-resistant gram-negative bacteria: a matched case-control study. Crit Case Med 2008; 36:807-811.. In three of the four cases reported herein, polymyxin B was administered immediately for over 14, 19, and 25 days, respectively, before the isolation of polymyxin-resistant and KPC-producing K. pneumoniae. The highest MIC was observed in an isolate from case 1, who received polymyxin for a longer period (25 + 14 days). The emergence of polymyxin B-resistant isolates has been observed during treatment with this drug for KPC-producing K. pneumoniae infection or colonization probably due to the selective pressure of polymyxin B on the heterogeneous bacterial population⁽¹³⁾13 Halaby T, Al Naiemi N, Kluytmans J, van der Palen J, Vandenbroucke-Grauls CM. Emergence of colistin resistance in Enterobacteriaceae after the introduction of selective digestive tract decontamination in an intensive care unit. Antimicrob Ag Chemother2013; 57:3224-3229.. An in vitro study of multidrug-resistant colistin-susceptible K. pneumoniae revealed colistin had a rapid bactericidal effect albeit a low post-antibiotic effect; bacterial regrowth was attributed to the heteroresistance phenomena, which was detected in 15 of 16 isolates⁽¹⁴⁾14 Poudyal A, Howden BP, Bell JM, Gao W, Owen RJ, Turnidge JD, et al. In vitro pharmacodynamics of colistin against multidrug-resistant K. pneumoniae. J Antimicrob Chemother2008; 62:1311-1318.. In case 1, the persistence of bacteremia during the first KPC-producing K. pneumoniae infection for up to 10 days of polymyxin B administration as well as rectal colonization for more than 100 days despite the therapeutic course with this antibiotic are suggestive of heteroresistance.

Other factors may be involved in the development of polymyxin resistance. In the present cluster patients had severe organic alterations and was invaded with catheters and drains. Three patients received immunosuppressants, and all four received broad-spectrum antibiotic therapy for more than 30 days, which favored infection with Gram-negative bacilli. Advanced age, a history of surgery, and the administration of monolactams and beta-lactams combined with beta-lactamase inhibitors are associated with colistin-resistant K. pneumoniae infection⁽¹²⁾12 Matthaiou DK, Michalopoulos A, Rafailidis PI, Karageorgopoulos DE, Papaionnou V, Nemi G, et al. Risk factors associated with the isolation of colistin-resistant gram-negative bacteria: a matched case-control study. Crit Case Med 2008; 36:807-811.. The previous colonization or infection with polymyxin-susceptible K. pneumoniae detected in three of the four cases was probably related to the development of strains resistant to these antibiotics.

Polymyxin-resistant K. pneumoniae strains may disseminate via horizontal transmission⁽⁵⁾5 Marchain D, Chopra T, Poque RM, Perez F, Huyer AM, Rudin S, et al. Outbreak of colistin-resistant, carbapenem-resistant, K. pneumoniae in metropolitan Detroit, Michingan. Antimicrob Ag Chemother 2011; 55:593-599.. In the present series, three patients were admitted to the hematology or bone marrow transplantation wards, which are close to each other and share some healthcare professionals. Dissemination of the extensively resistant bacterial strain probably occurred from case 1 to cases 2 and 3 via the actions of healthcare professionals or by undetected colonization of other patients. Regarding case 4, although the patient was admitted to a ward far from the others, she spent two days in the ICU where she had been admitted seven days after case 2 had left the unit; case 2 was probably already infected with the polymyxin-resistant strain, because she had a bloodstream infection by this microorganism eight days after leaving the ICU. Therefore, transmission to case 4 must have occurred in this unit.

Extensive antimicrobial resistance is not necessarily indicative of a high-virulence K. pneumoniae phenotype. Case 1 only exhibited mucosal colonization, while case 4 presented with fever and cellulitis around the venous catheter. However, the remaining cases had bloodstream infections, which contributed to mortality. Polymyxin-resistant and KPC-producing K. pneumoniae has caused more deaths than KPC producers susceptible to polymyxins⁽¹⁵⁾15 Capone A, Giannella M, Fortini D, Giordano A, Meledandu M, Ballardini M, et al. High rate of colistin resistance among patients with carbapenem-resistant K. pneumoniae infection accounts for an excess of mortality. Clin Microbiol Infect 2013; 19:e-23-e30..

The treatment of patients infected with KPC-producing K. pneumoniae is impaired by the limited number of effective antimicrobials, which include polymyxins, tigecycline, and aminoglycosides⁽³⁾3 Rapp RP, Urban C. K. pneumoniae carbapenemases in Enterobacteriaceae: history, evolution, and microbiology concerns. Pharmacotherapy 2012; 32:399-407.. In addition to being resistant to polymyxins, three of the four bacterial isolates studied herein were non-susceptible to tigecycline in vitro, exhibiting susceptibility only to amikacin. A similar susceptibility profile has been observed in other studies, suggesting a global trend toward even greater resistance of KPC clones⁽¹⁵⁾15 Capone A, Giannella M, Fortini D, Giordano A, Meledandu M, Ballardini M, et al. High rate of colistin resistance among patients with carbapenem-resistant K. pneumoniae infection accounts for an excess of mortality. Clin Microbiol Infect 2013; 19:e-23-e30..

The present findings suggest the isolation of polymyxin-resistant and KPC-producing K. pneumoniae is associated with previous colonization by polymyxin-susceptible and KPC-producing strains in patients submitted to prolonged antibiotic therapy and previous administration of polymyxin B. Furthermore, there is additional evidence of horizontal transmission of this extensively drug-resistant clone.

ACKNOWLEDGMENTS

We thank Lucia Helena Vitali for her help with the polymyxin susceptibility test.

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