ANTIMICROBIAL SUSCEPTIBILITY OF CLINICALLY SIGNIFICANT ISOLATES OF ENTEROBACTER SPP . , OBTAINED FROM PATIENTS , HOSPITALISED IN VARNA UNIVERSITY HOSPITAL DURING THE PERIOD 2014 – 2016

Purpose: Rapidly increasing antimicrobial resistance in medically important bacterial species from family Enterobacteriaceae is one of the most significant microbiological, clinical and epidemiological issues of modern medicine. The aim of this study is to investigate the antibiotic susceptibility of clinically significant isolates of Enterobacter spp., obtained from patients, hospitalized in University Hospital “Saint Marina” – Varna during the period 2014 – 2016. Material and methods: a total of 433 clinical isolates of Enterobacter spp. from blood cultures, urine and wound secretions were studied. The species identification was made by conventional, semi-automated (Crystal, BD) and automated systems (Phoenix, BD). The susceptibility to piperacillin/tazobactam (TZP), ceftazidime (CAZ), meropenem (MEM), gentamicin (Gm), amikacin (Ak), ciprofloxacin (CIP), levofloxacin (LVX), trimethoprime/ sulfamethoxazole (SXT) and tetracycline (Tet) was tested by disc-diffusion method and / or automated system Phoenix 100, BD. The results were interpreted according to EUCAST 2016 guidelines. Results: The resistance in the studied collection of isolates, shown in increasing order is as follows: Ak, 4.2% < LVF, 25.4% < TZP, 37.4% < Tet, 38.7% < SXT, 40% < CIP, 44.1% < Gm, 49.7% < CAZ, 57%. Meropenem demonstrated fully preserved activity. In the group of CAZ resistant isolates, the levels of antimicrobial resistance are: Ak, 5.7% < LVF, 42.9% < Tet, 52.4% < SXT, 60.3% < TZP, 64.4% < CIP, 84.6% < Gm, 86.2%. The rate of CAZ resistant Enterobacter spp. was 66.9% among the urine isolates, 61.9% among those from blood culture and 46.3% in the group of isolates from wound secretions. In the three mentioned groups of isolates, the lowest level of resistance was detected to Ak (1.6%; 4%; 6.9%). The isolates from wound and blood cultures demonstrated the highest level of resistance to Gm (60.3%, 42.9%) and the urine isolates – to Tet (60%) and CIP (56.9%). Conclusions: CAZ resistant Enterobacter spp. demonstrated significantly higher levels of resistance in comparison to the whole studied group especially to quinolones and aminoglycosides. The highest level of CAZ resistant Enterobacter spp. was detected in the group of urine isolates.


INTRODUCTION
Species from genus Enterobacter are ubiquitous bacteria, widely distributed in nature.They are part of the normal enteric flora of humans and animals but also can cause nosocomial infections predominantly in immunocompromized patients and patients from the ICUs [1,2].
Recently, a selected group of bacteria has been described by the acronym of ESKAPE, and they cause most of the healthcare-associated infections: Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter species.These bacteria are clinically important not only because are causative agents of nosocomial infections but also because are a paradigm for pathogenesis, transmission and resistance [3].Bacteria from genus Enterobacter are associated predominantly with bacteremia, pneumonia, urinary tract and intra-abdominal infections, endocarditis, skin and soft tissue infections [1].
Enterobacter species are with intrinsic resistance to aminopenicillins, amoxicillin/clavulanic acid, cefoxitin and first-generation cephalosporins because of the production of chromosomally encoded, inducible Amp C âlactamase [1,4,5].This enzyme does not hydrolyse carboxy-and ureidopenicillins, II, III and IV generation cephalosporins and carbapenems [4,5].During the last decades Enterobacter isolates, resistant to extended spectrum cephalosporins have been reported more frequently [1,4].Usually, these isolates are associated with the production of plasmid encoded Extended Spectrum Beta Lactamases (ESBLs) and/or derepressed production of Amp C enzymes [1,4].The ESBLs are most widely dessiminated, and cause hydrolyses to penicillins and all generations cephalospoirins [6,7].Such isolates of Enterobacter spp.demonstrate very often cross-resistance to other antimicrobial agents -aminoglycosides, quinolones, trimethoprim/sulphamothoxazole, etc. [8].The resistance to β-lactams, quinolones, aminoglycosides, trimethoprim/sulphamothoxazole is higher in the European, Asian and some countries in South America than in the USA and Canada [9,10].Very worrisome is the fact that worldwide carbapenem-resistant isolates of Enterobacter have been reported with increasing frequency [1,4].
The study aimed to investigate the antimicrobial susceptibility of a collection of clinically significant isolates of Enterobacter spp., obtained from patients, hospitalized in the University Hospital "Saint Marina" -Varna, Bulgaria during the period 2014 -2016.

MATERIALS AND METHODS
Between January 2014 and December 2016, a total of 433 clinically significant Enterobacter spp.isolates were collected from patients, hospitalized in different wards of the University Hospital "Saint Marina" in Varna, Bulgaria.
The isolates were recovered from various clinical specimens: urine samples -130; blood -126; wound secretions -177.Species identification was made by the semi-automated and automated systems Crystal (BD) and Phoenix 100 (BD).Antimicrobial susceptibility to set of antimicrobial agents (ceftazidime, meropenem, tetracycline, gentamicin, amikacin, trimethoprime/sulphometoxazole, ciprofloxacin, levofloxacin) was determined by disk -diffusion method and/or by the automated system Phoenix 100 (BD).The results were interpreted according to EUCAST 2016 guidelines [11].

RESULTS
The results from the antimicrobial susceptibilty testing are presented on Figures 1, 2 and 3.

DISCUSSION
During the last decade, the species from genus Enterobacter are among the fifth most commonly isolated bacterial species from clinical materials worldwide [2, 4].In Varna University Hospital "Saint Marina", Enterobacter cloacae takes the second place as a causitive agent of bacteremia for 2015 and the fourth place for 2016 (not published data).
In a recent study performed in our hospital in 2014, the ESBLs production in clinical isolates of Enterobacter spp., collected during the period 2007 -2011 from hospitalized patients was investigated.An increasing level of ESBL -producing Enterobacter spp. was detected: from 28% in 2007 ã. to 36% and 39% in 2010 and 2011 [15].This was followed by the current study where we detected 57% ceftazidime resistant isolates.This result shows the need for urgent infection control measures.
In the present study higher levels of resistance to all tested antimicrobials were detected in the group of ceftazidime resistant isolates.This result probably is associated with the possible production of plasmid mediated ESBLs or/and Amp C production.Different types of plasmids, identified in family Enterobacteriaceae, play an important role in the dissemination of ESBL genes [16,17].Some of these plasmids (IncFI, IncFII, Inc A/C, Inc M/L and IncHI2) carry not only ESBLs genes, but also genes, that encode plas-mid-mediated quinolone resistance (qnr, aac (6')-Ib-cr, qepA, oqxAB) and genes, associated with resistance to other antimicrobial agents (aminoglycosides, trimethoprime/sulfametoxazole) [6,9,18,19,20,21,22,23,24].This fact explains the common association of ceftazidime resistance (an indicator for ESBLs or/and AmpC production) with the resistance to other antimicrobial groups and the MDR phenotype, usually exhibited by these isolates.
Similar to the urine isolates, high levels of resistance to all tested antimicrobials were also detected in the group of blood culture isolates.Most of the ceftazidime resistant isolates from blood samples demonstrated multi-drug resistance.The high rate of resistance to extended spectrum beta-lactams, quinolones and aminoglycosides is a serious therapeutic challenge, as well as a significant factor for the outcome of blood stream infections caused by Enterobacter spp.In this study carbapenems and amikacin exhibit, the highest in vitro activity against Enterobacter spp., isolated from blood cultures with no resistance detected for carbapenems and 1.6% resistance for amikacin.Many authors report results that clearly demonstrate the advantage of carbapenem therapy for blood stream infections caused by ESBL -producing E. cloacae [27,28].
In all three groups of isolates (blood, urine and wound secretions) the most active antimicrobial agents were carbapenems with fully preserved activity and amikacin with resistance levels varying between 1.6%, 4% and 6.9%.
In conclusion, the studied collection of 433 isolates of Enterobacter spp. is associated with high levels of resistance to commonly used antimicrobial agents in the clinical practice: piperacillin/tazobactam, ceftazidime, quinolones and gentamicin.In comparison to the whole studied group, ceftazidime resistant Enterobacter spp.demonstrated significantly higher levels of resistance to most of the tested antimicrobials as well as MDR phenotype.The highest level of ceftazidime resistant Enterobacter spp. was detected in the group of urine isolates.
In the studied collection of isolates, the resistance to amikacin is the lowest, and the susceptibility to carbapenems is fully preserved.These antimicrobials are agents of the first choice in cases of severe infections caused by Enterobacter spp. or in cases of infections associated with MDR Enterobacter spp.