MOLECULAR EPIDEMIOLOGY OF MULTIDRUG RESISTANT ENTEROBACTER CLOACAE BLOOD ISOLATES FROM A UNIVERSITY HOSPITAL

urpose: to evaluate the epidemiological relationship between 3rd generation cephalosporin resistant Enterobacter cloacae blood isolates collected from patients in the University Hospital in Varna city during the period March 2014 and January 2017 and to characterize the ESBLs production in these isolates. Materials and methods: a total of 47 consecutive (nonduplicate) 3rd generation cephalosporin resistant isolates of Enterobacter cloacae, obtained from blood samples of patients admitted in different wards in Varna University Hospital, were investigated. Antimicrobial susceptibility to set of antimicrobial agents was tested by disc diffusion method and Phoenix (BD), and the results were interpreted according to EUCAST guidelines 2017. Identification of ESBL encoding genes was performed by PCR and sequencing. Isolates were genotyped by ERIC PCR. Results: The antimicrobial susceptibility in the whole collection of isolates, shown in decreasing order, is as follows: amikacin, 97.8% < levofloxacin, 76.6% < trimethoprime/ sulphometoxazole, 40.4% < ciprofloxacin, 19% < gentamicin, 8.4% < cefepime, 4.2% < piperacillin/ tazobactam, tobramycin, 2.1%. Multidrug resistance was detected in 70.2% of the isolates. The most widespread enzyme was CTX-M-15, found in 95.5% (n=43). Nine different ERIC types were detected. The dendrogram of similarity revealed three main clones of E. cloacae: Clone I, comprising two closely related subclones (ERIC type A and Aa) (similarity coefficient 0.92), was predominant, detected in Haematology (n=9), Haemodialysis (n=8), ICU (n=6), Cardio surgery (n=3), Pulmonology (n=4) and Gastroenterology (n=1); Clones II (ERIC type C) and III were presented by 5, and 3 isolates with identical profiles, obtained from patients, hospitalized in different wards. The ERIC profiles K, L, M and P, were found in single isolates only and were interpreted as sporadic. Conclusions: multi-drug resistance in E. cloacae was associated with successful intrahospital dissemination of three CTX-M-15 producing E. cloacae clones. Clone I was predominant, demonstrating high cross-transmission, epidemic and invasive potential. BlaCTX-M-15 was identified as a major mechanism of resistance to 3rd generation cephalosporins in E. cloacae

Microbiological and Molecular Genetic Studies on the Prevalence and Mechanisms of Resistance to Beta-Lactams and Quinolones in Clinically Relevant Enterobacter Spp. // Микробиологични и молекулярно-генетични проучвания върху разпространението и механизмите на резистентност към бета-лактами и хинолони при клинично значими Enterobacter spp.

This study aims to investigate the species composition, sensitivity and mechanisms of resistance to beta-lactams and quinolones in clinically significant isolates of Enterobacter spp., collected at St. Marina University Hospital - Varna for the period 2014-2017., as well as to determine the clonal relationship of the isolates. One hundred and seventy-five clinical isolates, isolated between March 2014 and January 2017 at St. Marina University Hospital - Varna, were investigated. The study also included an E. cloacae isolate from a hospital environment. The analysis of the results obtained in our research and its comparison with the literature gives us the reason to draw the following conclusions: 1) The examined isolates E. cloacae complex belonged to 99.2% of the species E. hormaechaei. Conventional identification methods, particularly the Phoenix100 automated system, do not have sufficient discriminatory power to distinguish the species in the E. cloacae complex. Hsp60 sequencing is a rapid and specific method for identification and distinguishing species in Enterobacter cloacae complex. 2) We found a high level of resistance (57%) to third-generation cephalosporins in the studied isolates as well as a high level of resistance to the most commonly used antimicrobial drugs in the clinical practice: piperacillin/tazobactam, ceftazidime, ciprofloxacin and gentamicin. Following the carbapenem imipenem and meropenem, the highest activity against Enterobacter spp. was amikacin. 3) The phenotypic methods for detecting ESBLs in clinical isolates of Enterobacter spp. showed unsatisfactory sensitivity. 4) Molecular genetic studies have shown resistance to third-generation cephalosporins among Enterobacter spp. isolates are due to the presence of ESBLs enzymes in over 85%, with the leading importance of CTX-M-15 for E. cloacae complex and CTX-M-3 ESBLs for E. aerogenes. In single isolates, we detected SHV-12 ESBL production. In non-ESBLs-producing isolates, resistance is most likely due to the hyperproduction of AmpC enzymes. Only one isolate has been shown to produce the DHA-1 enzyme. 5) Conjugation experiments demonstrated the plasmid localization of the ESBLs genes and PMQR and confirmed their contribution to the development of resistance to third-generation cephalosporins and selection of quinolone resistance. 6) The epidemiological study found extensive intra-hospital dissemination and long-term presence of one major clone (clone A) of E. cloacae complex with pronounced invasive potential. The microbiological study of the hospital environment proved E. cloacae complex identical (clone A, ERIC type Aa) with clinical isolates from different hospital units. 7) In 59% of Enterobacter spp. we found a presence of PMQR with qnrB dominance (90%) and a relatively low prevalence of qnrA, qnrS and aac (6') - Ib-cr (single isolates). QnrB was associated with CTX-M-15, qnrA - with SHV-12, and qnrS - with CTX-M-3. The only isolate with the qnrB4 allele produces CTX-M-3 and DHA-1. In over 50% of the isolates, we have shown chromosomal mutations for the gyrA and parC genes.Целта на настоящия дисертационен труд е да се проучат видовия състав, чувствителността и механизмите на резистентност към бета-лактами и хинолони при клинично - значими изолати Enterobacter spp., колекционирани в УМБАЛ’’ Света Марина’’ – Варна за периода 2014 - 2017 г., както и да се определи клоналната свързаност на изолатите. Във връзка с това бяха проучени сто седемдесет и пет клинични изолата, изолирани в периода март 2014 г.- януари 2017 г. в УМБАЛ '' Света Марина'', Варна. В проучването беше включен и един изолат E. cloacae от болнична среда. Анализът на резултатите, получени в нашите изследвания и съпоставката им с литературната информация ни дават основание да направим следните изводи: 1) Изследваните изолати Е. cloacae complex принадлежаха в 99.2% към вида E. hormachaei. Конвенционалните методи за идентификация и в частност автоматизираната система Phoenix100 няма достатъчна дискриминираща сила да разграничи видовете от E. cloacae complex. Нsp60 секвенирането е бърз и специфичен метод за идентификация и разграничаване на видовете в Enterobacter cloacae complex. 2) Установихме високо ниво на резистентност (57%) към цефалоспорини от III-та генерация в проучваната колекция от изолати, както и високо ниво на резистентност към най-често използваните антимикробни лекарствени препарати в клиничната практика: piperacillin/tazobactam, ceftazidime, ciprofloxacin и gentamicin. След карбапенемите imipenem и meropenem, с най - голяма активност срещу Enterobacter spp. се отличава amikacin. 3) Проведените фенотипни методи за детекция на ESBLs при клинични изолати Enterobacter spp. показаха незадоволителна чувствителност. 4) Чрез молекулярно-генетични изследвания доказахме, че резистентността към цефалоспорини от III-та генерация сред изолатите Enterobacter spp. се дължи на наличие на ESBLs ензими в над 85%, с водещото значение на CTX-M-15 за Е. cloacae complex и CTX-M-3 ESBLs за E. aerogenes. При единични изолати установихме продукция на SHV-12 ESBL. При изолатите без продукция на ESBLs резистентността се дължи най-вероятно на хиперпродукцията на AmpC ензими. Само при един изолат бе доказана продукция на DHA-1 ензим. 5) Конюгационните експерименти доказаха плазмидната локализация на ESBLs гените и PMQR и потвърдиха приноса им за развитие на резистентност към цефалоспорини от III-та генерация и селектиране на хинолонова резистентност. 6) Епидемиологичното проучване установи широка вътреболнична дисеминация и трайно присъствие на един основен клон (клон А) E. cloacae complex с изразен инвазивен потенциал. При микробиологичното изследване на болнична среда бе доказан изолат E. cloacae complex, генетично идентичен (клон А, ERIC тип Аа) с клинични изолати от различни клиники на болницата. 7) В 59% от изследваните изолати Enterobacter spp. установихме наличие на PMQR с доминиране на qnrB (90%) и сравнително слабо разпространение на qnrA, qnrS и aac(6')-Ib-cr (единични изолати). QnrB се асоциира с продукция на CTX-M-15, qnrA - с SHV-12, а qnrS - с продукция на CTX-M-3. Единственият изолат с алел qnrB4 продуцира CTX-M-3 и DHA-1. В над 50% от изолатите доказахме наличие на хромозомни мутации за gyrA и parC гените

Etiological spectrum of enteric infections in patients hospitalized in the university hospital `Saint Marina` - Varna

Цел: да се проучи етиологичния спектър и антибиотичната резистентност на най-честите причинители на инфекции на гастро-интестиналния тракт (ГИТ) в пациенти, хоспитализирани в инфекциозните и не-инфекциозни клиники на МБАЛ`Света Марина` - Варна в периода юли 2016 - февруари 2017г. Материали и методи: изследвани са общо 2165 фекални проби за най-честите бактериални и вирусни инфекциозни агенти: Salmonella spp., Shigella spp., Y. enterocolitica, V. cholerae (брой изследвани проби, n=2047), E. coli (n=1311), Campylobacter spp. (n=50), C. difficile (n=428), Rotavirus, Norovirus, Adenovirus и Astrovirus (n=560) чрез културелни методи за бактериалните причинители и имуно-хроматографски за вирусните, C. difficile и Campylobacter spp. Резултати: положителните фекални проби от общия брой изследвани материали са 19.9% (n=430). Вирусна етиология на инфекцията бе доказана в 66.7% от всички положителни материали (Rotavirus, 50%; Norovirus, 7.4%; Adenovirus, 7%; Astrovirus, 2.3%); делът на изолатите Salmonella spp., Shigella spp., ентеропатогенни E. coli и Campylobacter spp. от положителните фекални проби бе съответно 10.9%, 1.4%, 1.6% и 1.6%. От изследваните общо 428 материала за C. difficile, токсигенни щамове бяха идентифицирани в 17.8%, като съответно 16.6% (n=64) от фекалните проби от не-инфекциозни клиники (n=385) бяха положителни и 27.9% (n=12) от тези, получени от пациенти, хоспитализирани в Инфекциозните клиники на МБАЛ`Света Марина` (n=43). При 14 от пациентите във възрастта до 3г. бяха доказани случаи на коинфекции (Adenovirus + Astrovirus + Rotavirus + Norovirus; Astrovirus + C. difficilae + S. enteritidis; Rotavirus + Adenovirus + S. enteritidis и др.). Сред изолатите Salmonella spp. най-честият серотип бе S. enteritidis (n=29), следван от S. typhimurium (n=14). Резистентността на изолатите Salmonella spp., към антибиотици е съответно: pefloxacin - 4%; trimethoprim/sulfаmethoxazole - 8.5%; ampicillin - 40%. Заключение: с най - голямо етиологично значение за инфекциите на ГИТ са вирусните агенти, с водещото значение на Rotavirus за възрастта до 3г., следвани от C. difficile и Salmonella spp. Хинолоните и trimethoprim/sulfаmethoxazole са със съхранена активност срещу Salmonella spp.Aim: to study the etiological spectrum and antimi- crobial resistance of most frequently isolated infectious agents of enteric infections in patients, hospitalized in the infectious and non-infectious wards of University Hospital `Saint Marina` - Varna for the period July 2016 - February 2017.Мaterial and methods: a total of 2165 fecal sam- ples were tested for the most important bacterial and viral agents: Salmonella spp., Shigella spp., Y. en- terocolitica, V. cholerae (tested samples, n=2047), E. coli (n=1311), Campylobacter spp. (n=50), C. difficile (n=428), Rotavirus, Norovirus, Adenovirus and As- trovirus (n=560).Results: The positive fecal samples were 19.9% (n=430). Viral etiology was identified in 66.7% of all positive samples: Rotavirus, 50%; Norovirus, 7.4%;Adenovirus, 7%; Astrovirus, 2.3%; Salmonella spp., Shigella spp., EPEC and Campylobacter spp. were de- tected in 10.9%, 1.4%, 1.6% and 1.6% respectively. In the group of 428 samples tested for C. difficile, toxi- genic strains were identified in 17.8% (16.6% (n=64) from the samples, collected from the non-infectious wards (n=385) and respectively - 27.9% (n=12) from these, obtained from patients, hospitalized in the In- fectious wards of the Hospital (n=43). Co-infections were found in 14 patients in the age of 0 - 5 years: Ade- novirus + Astrovirus + Rotavirus + Norovirus; Astro-virus + C. difficile + S. enteritidis; Rotavirus + Adeno- virus + S. enteritidis, etc. Among Salmonella spp. iso- lates, S. enteritidis (n=29) and S. typhimurium (n=14) were the most common serotypes. The resistance rates of Salmonella spp. isolates to antimicrobial agents is as followes: pefloxacin - 4%; trimethoprim/sulfаmethox- azole - 8.5%; ampicillin - 40%.Conclusion: The viral pathogens were the most commonly identified etiological agents, with the lead- ing role of Rotavirus in the age of 0 - 5 years, followed by C. difficile and Salmonella spp. Quinolones and tri- methoprim/sulfаmethoxazole are with preserved ac- tivity against Salmonella spp

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: Аk, 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: Аk, 5.7% <LVF, 42.9% <Tеt, 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 Тet (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

First Report of DHA-1 Producing Enterobacter cloacae Complex Isolate in Bulgaria

The aim of the present study was to reveal the characteristics of an Enterobacter cloacae complex isolate producing DHA-1 AmpC enzyme recovered from a patient hospitalized in St Marina Hospital, Varna.Materials and methods: Susceptibility testing, conjugation experiments, isoelectric focusing, PCR and sequencing were carrying out.Results: Of 176 Enterobacter spp. isolates only one isolate was positive for blaDHA. The sequencing revealed the presence of blaDHA-1 and blaCTX-M-3. The antimicrobial susceptibility testing showed higher resistance rates to almost all beta-lactams (ceftazidime, cefotaxime, cefepime, amoxicillinclavulanic acid, piperacillin/tazobactam), tobramycin, gentamycin, trimethoprim/sulphomethoxazole and quinolones (ciprofloxacin and levofloxacin). The isolate was susceptible to imipenem, meropenem and amikacin. The isoelectric focusing showed a band at pI 5.4 without ceftazidime and cefotaxime activity; a band at pI 7.8 with cefoxitin activity and another - with pI 8.4 with cefotaxime activity. Conjugation experiments were successful only for blaCTX-M-3 carrying determinants. Conclusions: To the best of our knowledge this is the first report of DHA-1 producing isolate in Bulgaria. The emergence of DHA-1 producing E. cloacae complex demonstrates the possibility for further dissemination of the gene encoding this enzyme. Infectious control measures are needed for the prevention of this phenomenon

PYOGENIC LIVER ABSCESS - ETIOLOGICAL SPECTRUM AND SENSITIVITY TO ANTIBIOTICS

Introduction: Pyogenic liver abscess (PLA) is a serious challenge in modern medical practice. The aim of this study was to investigate the etiology and antimicrobial susceptibility of PLA-associated microbial pathogens, diagnosed in hospitalized patients at St. Marina University Hospital of Varna during the period between 2001 and 2016.Materials and Methods: A total of 84 clinical samples (pus aspirates, n=72, bile samples, n=7, and blood cultures, n=5), collected from PLA patients, hospitalized in the Second Surgery Clinic were analyzed. Species identification was performed by conventional methods. Antimicrobial susceptibility was studied by disk diffusion method and Phoenix 100 (BD). The results were interpreted according to CLSI and EUCAST standards.Results: Causative bacterial agents belonging to 15 different species were isolated in 59 cases (in 70%). E. coli (23.7%), K. pneumoniae (20%), E. cloacae (13.5%), E. faecalis (8.5%), P. mirabilis (5%) and P. aeruginosa (5%) dominated in the etiological spectrum. E. coli demonstrated the following levels of antimicrobial susceptibility: imipenem, amikacin, piperacillin/tazobactam, 100%; ceftazidime, cefepime, 90.9%; cefuroxime, 83.3%; amoxicillin/clavulanic acid, 77.8%; gentamicin, 75%; levofloxacin, cefalothin, 66.6%; ciprofloxacin, 63.6%; piperacillin, 58.3%; ampicillin, 45.5%. The following antimicrobial susceptibility rates were determined for K. pneumoniae: imipenem, 100%; amikacin, 92.3%; ceftazidime, cefepime, 80%; cefalothin, cefuroxime, 75%; levofloxacin, gentamicin, 66.6%; ciprofloxacin, 62.5%; piperacillin/tazobactam, 57.1%; piperacillin, 25% and amoxicillin/clavulanic acid, 22.2%.Conclusions: E. coli and K. pneumonia are the predominant pathogens in PLA patients. Carbapenems are the most active antimicrobial agents followed by ceftazidime and cefepime. In the aminoglycoside group, amikacin demonstrates the best in vitro activity