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ОРИГИНАЛЬНЫЕ ИССЛЕДОВАНИЯ
Антибиотикограмма штаммов Enterococcus spp., выделенных от пациентов с послеоперационной раневой инфекциеи
Шивашанкар Ш.Б.К., Бхат Н.Р., Дханашри Б.
Медицинский колледж Кастурба, Мангалор, Манипальская академия высшего образования, 575001, Манипал, Индия
При лечении пациентов в стационарных учреждениях здравоохранения из образцов раневого отделяемого все чаще выделяют энтерококки с множественной лекарственной устойчивостью, обладающие высокой резистентностью к аминогликозидам, р-лактамам и гликопептиду. Основными факторами риска развития устойчивости являются сниженный иммунный статус, длительное пребывание в стационарном лечебном учреждении, лечение антибактериальными препаратами широкого спектра действия, такими как цефалоспорины и ванкомицин.
Цель исследования - определение антибактериальной чувствительности штаммов Enterococcus spp., выделенных из образцов гнойного отделяемого больных с послеоперационной раневой инфекцией.
Материал и методы. В исследование включены представители 30 видов Enterococcus spp. Штаммы Enterococcus spp. были выделены из биологического материала пациентов с послеоперационными раневыми инфекциями. Изоляты были идентифицированы с помощью стандартных биохимических тестов. Продукцию гемолизина определяли путем посева штаммов Enterococcus spp. на 5% агар с овечьей кровью. Диско-диффузионный тест Кирби-Бауэра использовали для определения чувствительности к антибактериальным средствам. Результаты теста интерпретировали в соответствии с рекомендациями CLSI. Минимальную ингибирующую концентрацию ванкомицина (MIC) определяли методом разведения в агаре.
Результаты и обсуждение. Среди 30 изолятов энтерококка 18 (60%) идентифицированы как E. faeca-lis, по 4 (13%) были E. faecium и E. dispar, по 2 (7%) были E. pseudoavium и E. durans. 11 (61%) штаммов E. faecalis и 2 (7%) штамма E. faecium были положительными на гемолизин. 4 (22%) штамма E. faecalis были высокоустойчивы к стрептомицину. 8 (44%) штаммов E. faecalis и 1 (25%) штамм E. faecium показали устойчивость к гентамицину высокого уровня. Все изоляты энтерококка были чувствительны к ванкомицину при тестировании диско-диффузионным методом и разведении в агаре с MIC <2 мкг/мл.
Заключение. Своевременное выделение энтерококков и оценка их чувствительности к антибактериальным препаратам позволяет проводить эффективную этиотропную терапию, что способствует снижению летальности у госпитализированных пациентов и ограничивает распространение антибиотикорезистент-ных штаммов в больницах.
Ключевые слова:
антибактериальная
устойчивость; Enterococcus spp.; гемолизин; гной; раневая инфекция
Финансирование. Исследование не имело финансовой поддержки. Конфликт интересов. Авторы заявляют об отсутствии конфликта интересов.
Вклад авторов. Экспериментальная работа и обзор литературы для рукописи, анализ данных и написание текста - Шивашанкар Ш.Б.К.; экспериментальная работа, анализ данных и написание текста - Бхат Н.Р.; проект исследования, отслеживание экспериментов, анализ данных и рецензирование рукописи - Дханашри Б. Все авторы рассмотрели и одобрили окончательный вариант рукописи.
Благодарность. Авторы благодарят сотрудников кафедры микробиологии, декана Медицинского колледжа Кастурба (Мангалор) за поддержку в проведении исследования.
Для цитирования: Шивашанкар Ш.Б.К., Бхат Н.Р., Дханашри Б. Антибиотикограмма штаммов Еп1вгососсиз Брр., выделенных от пациентов с послеоперационной раневой инфекцией // Инфекционные болезни: новости, мнения, обучение. 2023. Т. 12, № 1. С. 84-89. 001: https://doi.org/10.33029/2305-3496-2023-12-1-84-89 (англ.) Статья поступила в редакцию 10.07.2022. Принята в печать 26.12.2022.
Antibiogram of Enterococcus spp. associated with post-operative wound infections
Shivashankar S.B.K., Bhat N.R., Department of Microbiology, Kasturba Medical College, Mangalore, Dhanashree B. 575001, Manipal Academy of Higher Education, Manipal, India
Multiple drug resistant enterococci with resistance to high level aminoglycoside, p-lactums, and glycopep-tide are increasingly being isolated from pus samples of hospitalised individuals. Risk causes include an immunocompromised state of the host, a prolonged hospital stay, and treatment with broad spectrum antibiotics like cephalosporins and vancomycin.
Aim: to study the antimicrobial susceptibility patterns and hemolysin production by various Enterococcus spp. isolated from infected surgical site wounds.
Material and methods. A total of 30 Enterococcus spp. isolated from post-surgery wound infections were included in the study. Standard bio-chemical tests identified and speciated them. The production of hemolysin by isolates was detected by inoculation of Enterococcus spp. on 5% sheep blood agar plate. To perform antibiotic sensitivity and interpret the results as per CLSI guidelines Kirby-Bauer's disc diffusion test was used. The Vancomycin Minimum inhibitory concentration (MIC) was detected by agar dilution method.
Results and discussion. Among the 30 enterococcal isolates, 18 (60%) were identified as E. faecalis, 4 (13%) were E. faecium, and E. dispar, 2 (7%) each were E. pseudoavium and E. durans. 11 (61%) E. faecalis and 2 (7%) E. faecium were haemolysin positive. 4 (22%) E. faecalis were resistant to High Level Streptomycin. 8 (44%) E. faecalis and 1 (25%) E. faecium showed resistance to High Level Gentamicin. All Entero-coccus isolates were susceptible to vancomycin by disc diffusion and agar dilution methods with an MIC of <2 |jg/mL.
Conclusion. Effective and appropriate identification and recognition of drug resistant Enterococcus spp. help in minimizing the morbidity and mortality in hospitalized patients and limit the spread of drug resistance in hospitals.
Funding. This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.
Conflict of interest. The authors declare that there are no conflicts of interest.
Contribution. Performed experimental work and background literature review for the manuscript, analysed data, and wrote the manuscript - Shivashankar S.B.K.; performed experimental work, analysed data, wrote manuscript - Bhat N.R.; designed the study, monitored the experiments, analysed data, and reviewed manuscript - Dhanashree B. All authors reviewed and approved the final version of the manuscript.
Acknowledgment. Authors thank the Staff of the Department of Microbiology, Dean, KMC Mangalore for their support to conduct the research.
For citation: Shivashankar S.B.K., Bhat N.R., Dhanashree B. Antibiogram of Enterococcus spp. associated with post-operative wound infections. Infektsionnye bolezni: novosti, mneniya, obuchenie [Infectious Diseases: News, Opinions, Training]. 2023; 12 (1): 84-9. DOI: https://doi.org/10.33029/2305-3496-2023-12-1-84-89 Received 10.07.2022. Accepted 26.12.2022.
Keywords:
drug resistance; Enterococcus spp.; haemolysin; Pus; surgical site infection
Enterococcus spp., once recognized as opportunistic pathogen, are now gaining importance as hospital acquired and community acquired infectious agent [1]. The two most common enterococcus species are Enterococcus faecalis and Enterococcus faecium. The most frequent infections caused by enterococci are urinary tract infections (UTIs), intra-abdominal and intra-pelvic abscesses or post-surgery wound infections, blood stream infections (BSIs) [2]. A major reason why these organisms survive in hospital environment is the intrinsic resistance to several commonly used antibiotics and perhaps more importantly, their ability to acquire resistance to all currently available antibiotics, either by mutation or by receipt of foreign genetic material through the transfer of plasmids and transposons [3].
Further, Enterococci with high level aminoglycoside resistance (HLAR), p-lactamase production, and glycopeptide resist-
ance, including vancomycin resistant enterococci (VRE), have emerged, posing a therapeutic challenge to clinicians due to the ease of acquiring infection [4, 5]. The enterococcal virulence factors include the cytolysins, aggregation substance, adhesions, extracellular superoxide (ESO), extracellular surface protein (ESP), haemolysin and gelatinase [6].
Intestinal colonization by drug resistant Enterococcus spp. is more common than clinical infection. These colonized patients can be a possible source for dissemination of organisms to hospital environment, healthcare workers and to other patients [7]. Moreover, repeated surgery of the gastrointestinal tract, prolonged hospitalization are some of the factors to promote infection by Enterococcus spp. [8]. In case of surgical site infection, the role of Enterococci spp. in causing infection is not supported by some scientists [9]. Few Indian studies report the role of enterococci in surgical site infection [10].
Hence, the present study is undertaken to know the different Enterococcus spp. involved in surgical site infection and their antibiotic susceptibility pattern.
Material and methods
Isolation of Enterococcus spp. from pus samples
In this time bound experimental study of six months duration 30 Enterococcus spp. isolated from post-operative pus samples received at the department of Microbiology belonging to a tertiary care hospital was included following non-random sampling method. Gram stain of clinical samples showing predominantly Gram-positive cocci in pairs along with pus cells were processed further for culture. Samples were inoculated onto blood agar and MacConkey's agar plates and incubated at 37 °C for 24 h.
Only non-repetitive and pure isolates of Enterococcus spp. were included in the study. Enterococcus spp. isolated from pus samples having other microorganism were excluded from the study. All 30 isolates belonged to inpatients admitted in the surgical wards for abdominal surgery. It consisted of 20 male and 10 female patients Their age ranged from 34 years to 70 years.
Species identification
Characteristic colonies on the blood agar and MacConkey's agar plates that are Gram positive cocci arranged in pairs were picked up and identified by conventional methods using the following phenotypic tests: catalase, motility, growth in 6.5% NaCl broth, bile esculin hydrolysis, tolerance to tellurite, fermentation of mannitol and arabinose [11].
Detection of haemolysin production
Haemolysin production was detected by inoculating enterococci on freshly prepared 5% sheep blood agar. Plates were incubated overnight at 37 °C in an incubator and evaluated after 24-48 h. A clear zone of beta haemolyses around colonies on blood agar is taken as positive [6].
Antibiotic susceptibility test
Antimicrobial susceptibility to ampicillin, penicillin, genta-micin, tetracycline, erythromycin, chloramphenicol, vancomycin, teicoplanin, ciprofloxacin, high level gentamicin, and highlevel streptomycin, is done by Kirby Bauer disk diffusion and interpreted as per CLSI guidelines [12]. ATCC E. faecalis 29212 strain was used as a control.
High level aminoglycoside resistance
Detection of high-level aminoglycoside resistance was performed by disk diffusion method using disks containing High level gentamicin (120 pg) and high-level streptomycin (300 pg). Results were read after incubation at 35 °C for 24 h and after 48 h for streptomycin. A zone diameter of 6 mm indicates resistance, 7-9 mm indicates inconclusive results and more that 10mm indicates susceptibility to aminoglycosides.
Resistance and susceptible to high-level gentamy-cin by disc diffusion method corresponds to MICs of >500 and <500 pg/mL respectively. For high-level streptomycin,
a zone diameter of 6 mm by disk diffusion corresponds to a MIC of >1000 pg/mL by broth dilution and >2000 pg/mL by agar dilution method. Moreover, for HLS a zone diameter of 10 mm by disk diffusion corresponds to a MIC of <500 pg/mL by broth and <1000 pg/mL by agar dilution method [13].
Determination of vancomycin minimum inhibitory concentration
The minimum inhibitory concentration (MIC) of vancomycin was determined by agar dilution method for all Entero-coccal isolates. Different concentrations of vancomycin were supplemented in BHI agar. Ten microliters of bacterial culture with the turbidity of McFarland 0.5 standard were spot inoculated. The plates were incubated at 37 °C for 24 h, examined for growth. The minimum concentration of vancomycin that suppressed the bacterial growth was taken as MIC. Enterococci ssp. with MIC >32 pg/mL were considered resistant; 8-16 pg/mL as intermediately resistant; and MIC of 4 pg/mL or less, as susceptible to vancomycin as per CLSI guidelines [13].
Results
Of the 30 enterococcal isolates, recovered from postoperative pus and wound swab, 18 (60%) were identified as E. faecalis, followed by 4 (13%) each as E. faecium, and E. dispar, 2 (7%) each as E. pseudoavium and E. durans (Fig. 1). 11 (61%) E. faecalis and 2 (50%) E. faecium, 1 (25%) E. dispar and 1 (50%) E. durans were p-haemolytic
(Fig. 2).
Among the total 30 enterococcal isolates, 11 were resistant to ampicillin, 8 to teicoplanin, 20 to erythromycin, 6 to chloramphenicol and 19 to cotrimoxazole, 5 to HLS and 12 HLG. All the isolates were susceptible to vancomycin by disc diffusion method and showed MIC of <2 pg/mL. 14 (46.6%) Enterococcus spp. were susceptible to both van-comycin and teicoplanin and 8 (27%) isolates were sensitive to vancomycin but resistant to teicoplanin. The antibiotic susceptibility pattern of different Enterococcus spp. is shown in the Table.
Of the 11 p-haemolytic E. faecalis 4 were resistant to HLS, HLG and teicoplanin. All the haemolytic E. faecalis were resistant to erythromycin. Among the 2 haemolysin producing isolates of E. faecium, 1 was resistant to teicoplanin and HLG. Both the isolates (100%) were resistant to ampicillin, erythromycin and cotrimoxazole. Amongst the 4 isolates of E. dispar, one was haemolysin positive, and was resistant to chloramphenicol, HLS, HLG, cotrimoxazole, ampicillin and erythromycin. Among the 2 isolates of E. durans 1 was haemolysin positive and was resistant to ampicillin, erythromycin, cotri-moxazole and teicoplanin.
Discussion
Vancomycin-resistant Enterococci have been reported from a wide variety of clinical samples worldwide. Extensive work on enterococci is being carried out because of their ability to cause serious infections and increase in resistance to many antimicrobial agents [14]. In the present study of 6 months
2.7%
2.7
4.13%
\
18.60%
4.13%
□ E. faecalis ■ E. faecium ■ E. dispar □ E. durans ■ E. pseudoavium
12 10 8 6
0
11
E. faecalis E. faecium E. dispar E. durans E. pseudoavium (n=18) (n=4) (n=4) (n=2) (n=2)
■ Haemolytic ■ Non-haemolytic
Fig. 1. Different Enterococcus spp. isolated from pus samples
Fig. 2. Haemolytic and non-haemolytic Enterococcus spp. isolated from pus samples
duration, about five different Enterococcus spp. were recovered from post-operative pus and wound swabs (see Fig. 1). ALL Enterococcus spp. isolated in this study were from patients who underwent operations of the upper or Lower gastrointestinal tract.
Previous study from Western India on post operative infections report the isolation rate of E. faecalis as 58%, and E. faecium to be 42% [15]. A similar study from Delhi, India also reports the isolation rate of E. faecalis to be 40%, E. faecium 56% and E. casseliflavus 4% [10]. Other studies from India have reported isolation of either E. faecalis, or E. faecium or both, but not other species [16, 17]. As depicted in figure 1, we have isolated five different Enterococcus spp., namely E. faecalis (60%) E. faecium (13%), E. dispar (13%) E. durans (7%) and E. pseudoavium (7%). Frequency of isolation of E. feacalis from skin and soft tissue infections was 12% from Egypt of which 53.8 % were VRE [18]. Thus, the distribution of Enterococcus spp. in post operative infection varies depending on the geographic area and the antibiotic steward ship practiced. Thus, our study emphasizes the importance of speciation of Enterococcus in clinical diagnostic Laboratory as we couLd isolate five different species.
ViruLence factor Like haemoLysin pLays an important roLe in pathogenesis of the disease [6]. HaemoLysin producing strains have been found to be viruLent in both animaL modeLs and human infections. HaemoLytic strains are known to be associated with higher severity of infection in humans [19]. We have characterized aLL 30 enterococcaL isoLates for haemoLysin production. It was observed that eLeven (61%) E. faecalis, 2 (50%) E. faecium, 1 (25%) E. dispar and one (50%) E. durans were p-haemoLytic (see Fig. 2). EarLier studies from PoLand and India showed that 30% of the E. faecalis to be haemoLytic [19, 20]. However, in the present study it is reported to be 61%. These p-haemoLytic enterococci were found to be resistant to more than three antibiotics (muLti drug resistant) incLuding HLS and erythromycin. EarLier study from north India reports isoLation of geLatinase and haemo-Lysin producing muLtidrug resistant Enterococcus spp. [21]. Thus, our resuLts agree with the earLier studies from India and abroad.
In the present study, among the 30 EnterococcaL isoLates, 37% were resistant to ampiciLLin, 27% to teicopLanin, 67% to erythromycin, 20% to chLoramphenicoL, 63% to cotrimoxa-zoLe, 17% to HLS and 30% to HLG. ALL the isoLates were susceptibLe to vancomycin by disc diffusion method, with MIC of <2 Mg/mL by agar diLution method. 14 (46.6%) Enterococcus spp. were susceptibLe to both vancomycin and teicopLa-nin and 8 (27%) isoLates were sensitive to vancomycin but resistant to TeicopLanin (see TabLe). Our resistance rates are Lower when compared to the study from Chennai which reports 82% resistance to erythromycin, 44% to HLG and 22% to HLS [17]. However, their study incLuded onLy E. faecalis and E. faecium. Lower rate of resistance seen in our isoLates is an indication of a strict adherence to infection controL practices and isoLation of different Enterococcus species other than E. feacalis & E. faecium.
In the present study 100% of the isoLates were susceptibLe to vancomycin. A study from west BengaL aLso reports isoLation of muLtidrug resistant enterococci susceptibLe to vancomycin from cLinicaL sampLes [22]. A study from Tehran reports 20% of their isoLates to be VRE [23]. A ten-year surveiLLance study from Germany reports an increase in VRE from 0.9 to 5.2%. These VRE are associated with bLood stream and urinary tract infections but not with surgicaL site infection [24]. Purohit et aL reports the VRE isoLation rate from pus sampLes to be 9.5% [16]. Thus, our hospitaL needs to be vigiLant for the deveLopment of vancomycin resistance in near future.
Rajkumari et aL reports that factors Like prior ICU stay, muLtipLe surgicaL interventions and proLonged hospitaL stay may Lead to deveLopment of drug resistance in enterococci which may predispose for surgicaL site infection [10]. CoLonization of intestine with drug resistant enterococci in patients act as a possibLe source for dissemination of organisms to hospitaL environment, heaLthcare workers and to other patients [7]. Moreover, repeated surgery of the gastrointestinaL tract, proLonged hospitaLization is some of the factors to promote infection by Enterococcus spp [8]. Thus, aLL previous findings show, isoLation of drug resistant or susceptibLe enterococci from surgicaL site infection depends on various factors Like intestinaL
Antibiogram of various Enterococcus spp. isolated from pus samples, n (%)
Antibiotics 1 E. faecalis (n=18) | 1 E. faecium (n=4) | 1 E. dispar (n=4) 1 E. durans (n=2) 1 E. pseudoavium (n=2) |
tested S R S R S R S R S R
Ampicillin i3 (72.2) 5 (27.В) 2 (50.0) 2 (50.0) 2 (50.0) 2 (50.0) 1 (50.0) 1 (50.0) 1 (50.0) 1 (50.0)
Erythromycin ¡7 (3В.9) ii (6i.i) 0 4 (i00.0) i (25.0) 1 3 (75.0) 1 (50.0) ¡I 1 (50.0) 1 (50.0) 1 1 (50.0)
Chloramphenicol i4 (77.В) 4 (22.2) 4 (i00.0) 0 3 (75.0) 1 (25.0) 2 (100.0) 0 1 (50.0) 1 (50.0)
Co-trimoxazole 1 5 (27.В) i3 (72.2) 2 (50.0) 2 (50.0) 2 (50.0) 1 2 (50.0) 1 (50.0) ¡I 1 (50.0) 1 (50.0) I 1 (50.0)
HLS* i4 (77.В) 4 (22.2) 4 (i00.0) 0 3 (75.0) 1 (25.0) 2 (100.0) 0 2 (100.0) 0
HLG* |l0 (55.6) В (44.4) 3 (75.0) i (25.0) 2 (50.0) 1 2 (50.0) 2 (100.0)|| 0 2 (100.0)|| 1 (50.0)
Teicoplanin i3 (72.2) 5 (27.В) 3 (75.0) i (25.0) 4 (i00.0) 0 1 (50.0) 1 (50.0) 1 (50.0) 1 (50.0)
Vancomycin i8 (i00.0) 0 4 (i00.0) 0 4 (100.0)| 0 2 (100.0)|| 0 2 (100.0)|| 0
S - susceptible; R - resistance.
colonization, dissemination to the environment, infection control practices and many more. In case of surgical site infection, the role of enterococci in causing infection is not supported by some scientists [9]. However, in the present study we could not get the details of predisposing factors like duration of hospitalization, ICU stay and history of prior antibiotic therapy of the patients which would have helped us to know the role of Enterococcus spp. in surgical site infection/colonization. This is one of the limitations of our study.
Conclusion
Our study revealed varying degrees of antibiotic resistance in five different species of Enterococcus isolated from pus. Isolation of drug resistant Enterococcus spp. is a concern as
resistance can be transferred to other bacterial species leading to limited treatment options. Species level identification of Enterococcus is not only important for epidemiological study, but also for analysing the drug resistance pattern. Treating Surgeon and microbiologist should be aware of the management of these neglected Enterococcal species mainly to tailor the antibiotic prophylaxis. To understand the role of virulence factors in pathogenesis of surgical wound infection, further studies are necessary to characterize a greater number of other Enterococcus spp.
Ethical approval. All procedures done in the study were in agreement with the ethical standards of the institutional and/ or national research committee. This study has been approved by Institutional Ethics committee Kasturba Medical College, Mangalore.
АВТОР ДЛЯ КОРРЕСПОНДЕНЦИИ
Дханашри Бирантабаил (Biranthabail Dhanashree) - PhD, кафедра микробиологии Медицинского колледжа Кастурба, Ман-галор, Манипальская академия высшего образования, Манипал, Индия E-mail: [email protected] https://orcid.org/0000-0002-5370-0663
CORRESPONDING AUTHOR
Biranthabail Dhanashree - Associate Professor, Department of Microbiology, Kasturba Medical College, Mangalore, Manipal Academy of Higher Education, Manipal, India E-mail: [email protected] https://orcid.org/0000-0002-5370-0663
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