Carbapenemase production in hospital isolates of multidrug-resistant Klebsiella pneumoniae and Escherichia coli in Serbia Produkcija karbapenemaza kod bolni č kih multirezistentnih sojeva Klebsiella pneumoniae i Escherichia coli u Srbiji

Background/Aim. Carbapenem resistance has escalated in medically important enterobacteria such as Klebsiella pneumoniae and Escherichia coli worldwide. Multidrug-resistant strains represent an important source of concern as effective therapeutic options of infections they cause are limited or none. There were no comprehensive studies considering the presence of carbapenemase production in enterobacteria in Serbia so far. The aim of the study was to determine carbapenemase production in hospital isolates of multidrug-resistant K. pneumoniae and E. coli in Serbia. Methods. Strains of K. pneumoniae and E. coli resistant to at least one carbapenem (imipenem, meropenem, ertapenem) were collected from November 2013 to May 2014. Isolates were obtained from clinical samples of patients treated in 14 hospitals in Serbia. Carbapenem resistance was confirmed using phenotypic tests and polymerase chain reaction (PCR) in National Reference Laboratory for Registration and Surveillance of Antimicrobial Resistance of Bacterial Strains in Novi Sad. Results. Of 129 collected strains, 121 (93.8%) were K. pneumoniae and 8 (6.2%) were E. coli. Seventy (54.3%) strains were obtained from urine, 26 (20.2%) from blood, 19 (14.7%) from wound secretions and 14 (10.9%) from lower respiratory tract secretions. Carbapenemase genes were detected in 58 (45%) isolates. The gene bla New Delhi-metallo-beta-lactamases (blaNDM) was found in 33 (27.3%) K. pneumoniae, bla oxacillinases-48 (blaOXA-48) in 10 (8.3%), bla K. pneumonia carbapenemase (blaKPC) in 1 (0.8%), and 7 (5.4%) strains harbored both blaOXA-48 and blaNDM. Seven E. coli harbored blaNDM gene. Conclusion. In Serbia, the most common type of carbapenemase in both multidrug-resistant K. pneumoniae and E. coli is NDM. Co-production of OXA-48 and NDM was found in K. pneumoniae. To our knowledge, KPC production was detected for the first time in Serbia.


Introduction
Due to increased global transport, there is an increased exposure of people all around the world to diverse Gramnegative bacteria from gut flora, especially Escherichia coli and Klebsiella spp.Fecal carriage is recognized as the most important for spreading multidrug-resistant strains in the hospital environment 1 .Multidrug-resistant bacteria represent an important source of concern as effective therapeutic options of infections they cause are limited or none 2 .Carbapenems are often used for the treatment of nosocomial infections as the last line therapy 3 .In the last decade, carbapenem resistance has escalated in medically important bacteria 4,5 .In Europe Klebsiella pneumoniae is the most frequently reported carbapenem-resistant enterobacteria 6 .Isolation of carbapenem-resistant E. coli is of concern, as it spreads more easily in the community.Also, the treatment of such community-acquired infections might become a challenge 7 .Two main mechanisms are responsible for carbapenem resistance, the first refers to carbapenem-hydrolyzing enzymes (carbapenemases) and the second is usually a combination of deficiency of porin expression and overexpression of betalactamases with weak affinity for carbapenems 7 .The most frequently isolated carbapenemases are K. pneumoniae carbapenemases (KPC), Verona integron-encoded metallo-betalactamases (VIM), imipenemases (IMP), New Delhi metallobeta-lactamases (NDM) and oxacillinases-48 (OXA-48) 5 .Carbapenemase-encoding genes are usually located on selfconjugative plasmids often accompanied with other nonbeta-lactam resistant determinants 8 .Acquisition of genetic material through horizontal transfer explains the urge for proper detection of carbapenemase-producing strains 7,8 .Unfortunately, the detection of carbapenemase producer cannot rely only on the resistance profile routinely done in microbiology laboratory as their minimal inhibitory concentration (MIC) values may sometimes lay within the susceptibility range.Also, some strains may produce other enzymes and mechanisms responsible for lower resistance to carbapenems 8,9 .Therefore, multidrug-resistant isolates with lower susceptibility to carbapenems should be tested for the presence of carbapenemase in order to prevent hospital outbreaks.To our knowledge, there were no comprehensive studies considering the presence and the occurrence of carbapenemase production in enterobacteria in Serbia so far.
The aim of the study was to determine carbapenemase production in hospital isolates of multidrug-resistant K. pneumoniae and E. coli in Serbia.

Methods
The study included 129 nonrepetitive multidrugresistant strains of K. pneumoniae and E. coli isolated from a clinical specimen (urine, blood, wound secretion/swab and lower respiratory tract secretions: tracheal aspirate, bronchoaspirate, broncho-alveolar lavage) from November 2013 to May 2014.The strains were collected from microbiology laboratories in Clinical Center Serbia (Belgrade), Clinical Center "Zvezdara" (Belgrade), Clinical Center "Dragiša Mišo-  10 .
The study was conducted at National Reference Laboratory for Registration and Surveillance of Antimicrobial Resistance of Bacterial Strains in the Institute for Public Health of Vojvodina, Novi Sad, Serbia.Identification of isolated strains was done using VITEK 2 Compact GN cards (BioMérieux, Marcy I Etoile, France).Antimicrobial susceptibility was determined using the disk diffusion method and/or using VITEK 2 AST-GN71 and AST-N240 cards according to CLSI.Susceptibility to fosfomycin was tested by E test strip (AB, Biodisk, Solna, Sweden).For the interpretation of tigecycline MICs, Food and Drug Administration (FDA) breakpoints for Enterobacteriaceae were used (susceptible ≤ 2 mg/L, intermediate 4 mg/L; resistant ≥ 8 mg/L).Phenotypic testing of extended-spectrum beta-lactamases (ESBL) production was done using combined disk tests (CDT) using cefotaxime disk and cefotaxime/clavulanic acid disk and ceftazidime and ceftazidime/clavulanic acid disk (Bio-Rad, France).Enhancement of the zone of inhibition more than 5 mm of the inhibitor-containing disc was considered to be a positive result.Phenotypic testing of carbapenemase production was done by double-disk synergy test (DDST) using tablets containing meropenem (10 µg), cloxacillin, dipicolinic acid, boronic acid (Rosco Diagnostica Neo-Sensitabs, Taastrup, Denmark) according to manufactures' instructions.Enhancement of the zone of inhibition in the area between meropenem disc and the inhibitorcontaining disc was considered to be a positive result.Confirmation of carbapenemase production was done using polymerase chain reaction (PCR).PCR reaction of five genes was performed as two separate multiplex reactions and one simplex reaction with Mastercycler personal (Eppendorf, Hamburg, Germany).The first reaction included primers for bla NDM 11 and bla KPC 12 genes, the second included primers for bla OXA48 13 and bla VIM 14 genes.PCR cycling conditions for the first reaction were 1 cycle at 95 ºC for 5 min, 30 cycles of 95 ºC for 30 s, 60 ºC for 30 s, and 72 ºC for 60 s, followed by 1 cycle at 72 ºC for 3 min and holding stage at 4ºC.PCR cycling conditions for the second reaction were 1 cycle at 95 ºC for 5 min, 30 cycles of 95 ºC for 30 s, 58 ºC for 30 s, and 72 ºC for 60 s, followed by 1 cycle at 72 ºC for 3 min and holding stage at 4 ºC.Gene bla IMP 14 was tested separately under following conditions: one cycle at 95ºC for 5 min, 30 cycles of 95 ºC for 30 s, 58 ºC for 30 s, and 72 ºC for 60 s, followed by 1 cycle at 72 ºC for 3 min and holding stage at 4ºC.All primers are shown in Table  The statistical analysis of the results was performed using the Statistical Package for the Social Sciences (SPSS), version 20.Results were expressed through the descriptive statistics, as simple frequencies and percentages.The χ 2 test was used for determination of statistically significant differences.The tested significance level was α = 0.05.

Results
The study included 129 isolates of multidrug-resistant K. pneumoniae and E. coli isolated from a different clinical specimen of hospitalized patients.There were 121 isolates of K. pneumoniae and 8 isolates of E. coli.The patients from whom the isolates were obtained included 69 (53.5%) males and 59 (45.7%) females.The patients' mean age was 53 (SD ± 24) years.According to the location in hospital in the when the sample was taken 71 (55%) were collected from non-intensive care units and 58 (45%) were taken from intensive care units.The distribution of clinical specimen is shown in Table 2.The antimicrobial resistance patterns of collected isolates are presented in Table 3.
Susceptibility to carbapenems of K. pneumoniae and E. coli is shown separately in Table 4.
pneumoniae and E. coli are shown in Table 5. Phenotypic tests for ESBL producers and carbapenemase producers were performed in order to enlighten the betalactam resistance mechanism of carbapenem-resistant strains.The results are presented in Table 6.
According to the hospital location in the moment of sampling 37 (63.8%)carbapenemase-producing isolates were collected from patients treated in intensive care units and 21 (29.6%) from other wards (χ 2 = 15.848;p = 0.007).

Discussion
K. pneumoniae and E. coli are frequently responsible for numerous community and hospital acquired infections 15 .Although originally being human commensals susceptible to almost all antimicrobial agents, in last 15 years we witness a rapid dissemination of multidrug-resistant strains 16 .Resis-tance to carbapenems is usually a consequence of the acquisition of carbapenemase genes.Being mostly plasmidencoded, carbapenemase-producing enterobacteria are often accompanied by other resistance genes 15 .The horizontal genetic transfer may cause rapid and extensive dissemination of multidrug-resistant carbapenemase-producing strains not only in healthcare facilities but also within the region or even across borders 17 .
On the other hand, carbapenemase-producing enterobacteria are not necessarily clinically resistant to carbapenems thus representing a diagnostic challenge to routine laboratories.Usually, recommendations are either based on epidemiological cutoff values of minimal inhibitory concentrations or clinical breakpoints for carbapenems.It is advised that screening criteria may and should be adjusted depending on the epidemiological situation in a given ecological setting 16 .So far, there have been no comprehensive studies considering the presence and the occurrence of carbapenemase production in enterobacteria in Serbia.Their presence may be missed if different criteria are followed, especially by laboratories lacking the experience in interpreting and performing phenotypic tests.A total of 129 multidrug-resistant strains with lower susceptibility to routinely used carbapenems was collected in 6 months period from various hospitals in Serbia.K. pneumoniae and E. coli were isolated mostly from urine and less frequently from blood, wound secretion or swab and lower respiratory tract secretions.Resistance rates for 13 tested antimicrobial agents were very high.Good activity maintained for amikacin, fosfomycin, tigecycline, and colistin.
Collected strains were all intermediately susceptible or resistant to ertapenem.Ertapenem is considered the most sensitive indicator for carbapenemase production, though often with low specificity due to either production of ESBL or overproduction of AmpC beta-lactamases 18 .In our study, none of solely ertapenem intermediate or resistant isolates harbored carbapenemaseencoding genes nor were positive in phenotypic testing for carbapenemase production.All isolates with carbapenemase genes were resistant to meropenem, suggesting that meropenem susceptibility might be an indicator for carbapenemase production among K. pneumoniae and E. coli in Serbia.
After performing phenotypic testing, 46 strains were suspected for carbapenemase production and 41 carried tested carbapenemase genes (bla KPC , bla NDM, bla VIM, bla IMP, bla OXA-48) .Positive phenotypic test in 5 isolates that tested negative for carbapenemase genes indicated the presence of metallo-beta lactamase.Additional testing is needed to detect the type of metallo-beta lactamase.
Phenotypic tests for carbapenemase production failed to detect 17 isolates with carbapenemase genes.There are no specific inhibitors for OXA-48 carbapenemase commercially available, therefore 7 strains would be missed without molecular testing.Also, phenotypic tests for carbapenemase production are unreliable when two different mechanisms of carbapenemase production occur 19 .
More isolates with carbapenemase production were found among samples collected in intensive care units compared to other wards.Together with various risk factors such as age of patients, underlying illness and co-morbid conditions of the host, the intensive care unit stay and carbapenem resistance are the most important predictors of increased mortality and treatment failure 16 .
Carbapenemase genes were detected in 58 isolates.The most prevalent carbapenemase was NDM found in both K. pneumoniae and E. coli.NDM producing strains are found in 9 hospitals from 7 different cities in Serbia.NDM was first identified in 2008 from K. pneumonia in Sweden from a patient treated in India 20 and has been reported worldwide 4 .India is considered to be endemic although after the first comprehensive analyses a smaller percentage of cases were connected to Balkan countries 4 .However, the most Balkan countries were lacking data or were uncertain considering the occurrence of carbapenemases among enterobacteria 6 .In Serbia, NDM was detected for the first time in Pseudomonas aeruginosa in 2010 21 .In 2011 NDM was detected for the first time in K. pneumoniae in Belgrade isolated from urine of a 7-month-old baby boy, though without any clinical signs of infection 22 .As for Bulgaria, an outbreak caused by NDM producing E. coli was reported 23 , but also VIM and KPC producing K. pneumoniae were documented showing the diversity of circulating carbapenemases 24 .In Croatia, VIM producing enterobacteria were the most prevalent but NDM producing strains were isolated 25 .In Greece, KPC and VIM reached epidemic proportions 6,26 .No available data were found considering Albania, Bosnia and Herzegovina and the Republic of Macedonia.NDM producing enterobacteria originated from Montenegro and Kosovo, Serbia was reported in Belgium 27 .Also, NDM and OXA-48 carbapenemase were found in carbapenem-resistant enterobacteria in the neighboring Romania 28 .In general, NDM did not reach such a wide distribution in Europe as KPC according to data from 2013 6 .United Kingdom has been reporting more NDM isolates comparing to other European countries 17 .Although an outbreak caused by metallo-lactamase producing Proteus mirabilis (VIM, IMP) in surgical intensive care unit of Clinical Center Serbia, Belgrade was described 29 , none of tested isolates carried bla VIM nor bla IMP genes.
OXA-48 was first detected in K. pneumoniae in Turkey followed with hospital outbreaks across the country 30 .Among European countries, OXA-48 was the most frequently detected in Belgium, France and Malta 6 .OXA-48 producing K. pneumoniae was reported in Slovenia 31 , but no OXA-48 carbapenemase was found in a multicentre study in Croatia 25 .Recently, in Hungary, two isolates of K. pneumoniae harboring OXA-48-like carbapenemases were characterized 32 .In our study OXA-48 carbapenemase was found in 10 K. pneumoniae isolates obtained from 4 different healthcare facilities from 3 different cities (Belgrade, Niš and Kikinda).
Co-production of OXA-48 and NDM in K. pneumoniae is rarely reported.According to published data, both OXA-48 and NDM were found in K. pneumoniae isolates in Tunisia 33 , Morocco 34 and Turkey 35 .An extensively drug-resistant isolate of K. pneumoniae with both OXA-48 and NDM obtained from a rectal swab of a patient transferred from the intensive care unit of a hospital located in Belgrade (Serbia) to Bern University Hospital in Switzerland was described 36 .Among collected isolates, both OXA-48 and NDM were found in 7 K. pneumoniae isolates.Isolates were obtained from patients hospitalized in 2 healthcare facilities in Belgrade from different clinical specimens (urine, wound secretion and blood).
The first KPC producing K. pneumoniae was identified in 1996 in the eastern part of the United States and has been spreading since 7 .KPC is the most frequently detected carbapenemase among Enterobacteriaceae in Europe particularly in Italy and Greece 6 .KPC harboring K. pneumoniae has been isolated in the neighboring Hungary 37 and Croatia 25 .In our study, KPC carbapenemase was detected in only one isolate of K. pneumoniae from the patient treated in the intensive care unit in the Institute for Pulmonary Diseases of Vojvodina near Novi Sad without previous hospitalization.As far as we know, KPC production was detected for the first time in Serbia.

Conclusion
To our knowledge, this is the most comprehensive national report on carbapenemase-producing enterobacteria in Serbia.NDM carbapenemase is the most prevalent among isolates of K. pneumoniae and E. coli.Also, rarely described co-production of OXA-48 and NDM is found in K. pneumoniae isolates.KPC production is documented for the first time.Further characterization of detected genes as well as further detailed epidemiological studies are needed.It is of great importance to make a unique and precise guideline for routine microbiology laboratories in order to detect carbapenemase-producing strains adequately and to monitor the epidemiological situation on the national and international level.