1. EXTENDED SPECTRUM β-LACTAMASE PRODUCING GRAM NEGATIVE BACTERIA ISOLATED FROM INFECTED WOUND OF ORTHOPEDIC PATIENTS Shwe Phyu Aung , Thandar Win Maung, San Mya, Wah Win Htike , Eh Htoo Pe , Ohnmar Aung

2. INTRODUCTION

3. Orthopedic wound infections
one of the most common hospital acquired infections
nowadays, gram negative orthopedic wound infection represents major proportion of occurrences orthopedic wound infection

4. In Turkey, 72 (75%) gram negative bacteria were isolated from orthopedic wound infection
the most common isolated bacteria were
Acinetobacter spp. (25%)
Pseudomonas aeruginosa (20%)
Escherichia coli (10%)
Klebsiella pneumonia (10%)

5. With the rapidly rising antimicrobial resistance rate among gram negative bacteria
the prevalence of Extended Spectrum β lactamase (ESBL) producing gram negative bacteria mainly among Enterobactericeae family is increasing all around the world.

6. In Thailand in 2012 revealed a high prevalence of multidrug resistance gram negative bacteria (48.8 %)
Among multidrug resistance gram negative bacteria, 37.8 % were ESBL producers (Suwantarat et al., 2016)

7. In Myanmar
Myat et al., (2014) observed that among 42 gram-negative, 16 (38%) were ESBL producing Enterobacteriaceae
Lei-Lei-Phyo (2017) showed that among 65 gram negative isolates, 13 (20%) isolates were ESBL producers. ESBL production was commonly seen in Escherichia coli (4/13, 30.77%).

8. This study also represents a local data of ESBL producing gram negative bacteria causing wound infections in Yangon Orthopedic Hospital
Thus, this research provides many beneficial effects for both clinicians and patients

9. AIM AND OBJECTIVES

10. AIM
To study the Extended Spectrum β-lactamases producing Gram negative bacteria isolated from infected wound of orthopedic patients

11. OBJECTIVES
To detect the proportion of Gram negative bacteria infection from infected wound of orthopedic patients
To determine the antibiotic susceptibility pattern of the Gram negative bacterial isolates
To detect the Extended Spectrum β-lactamases producing aerobic Gram negative bacteria among these isolates by double disc synergy test and phenotypic confirmatory test (cephalosporin/clavulanate combination disc diffusion method)

12. MATERIALS AND METHODS

13. The study is
a cross-sectional descriptive study
from January 2018 to October 2018
at Yangon Orthopedic Hospital
Bacteriology section, National Health Laboratory
Wound swabs from 151 patients who have any one of clinical signs of wound infection with or without prior antibiotic were collected.

14. Flow chart of the study
Collection of wound swabs after getting informed consent
Transport the wound swabs with Stuart’s transport medium to NHL (Bacteriology section) within 6 hours after collection
Isolation of bacteria by inoculation the specimen on Blood and MacConkey agar and incubated at 37° C overnight
Microscopic examination of specimen by direct smear
Identification of bacteria by inspecting colonial morphology and microscopic examination of
Gram stained smear

15. Gram positive bacteria Gram negative bacteria
Identify by API 20 E if GNB could not be identified by conventional method
Identify by conventional
method
Antibiotic susceptibility testing by modified
Kirby Bauer disc diffusion method
on Mueller-Hinton agar

16. Resistant to ceftriaxone, ceftazidine and cefotaxime
Suspect ESBL producing
Detect ESBL producing Gram negative bacteria by Double Disc Synergy Test and
Phenotypic Confirmatory Test (cephalosporin/clavulanate combination disc diffusion method).

17. Biochemical reactions of Klebsiella pneumoniae
Biochemical identification of Proteus mirabilis by API 20 E method

18. Antimicrobial susceptibility pattern of Klebsiella pneumonia on Mueller Hinton agar showing the suspected ESBL
CTX = Cefotaxime
CRO = Ceftriaxone
CAZ = Ceftazidime
AK = Amikacin
TZP = Piperacillin-tazobactam
MEM = Meropenem

19. ESBL production on Mueller Hinton agar by double disc synergy test
AMC = Amoxicillin-clavulanic acid
CTX = Cefotaxime
CAZ = Ceftazidime

20. Mueller Hinton agar plate showing ESBL production by combination disc test
CAZ = Ceftazidime
CAZ/CLA = Ceftazidime/clavulanic acid
CTX = Cefotaxime
CAZ/CLA = Cefotaxime /clavulanic acid

21. RESULTS

22. Total 151 wound swab samples Gram negative bacteria 103(67.76%)
Pathogens isolated samples
119 (78.8%)
Sterile
19 (12.6%)
Others
(Yeast, Gram positive bacilli)
13 (8.6%)
Total pathogens
152
Gram positive bacteria
49 (32.24%)
Staphylococcus aureus – 23
CONS – 15
Streptococcus spp. – 11
Gram negative bacteria 103(67.76%)
Non-ESBL producers
(90, 87.38%) isolates
ESBL producers
(13, 12.62%) isolates

23. Distribution of study population by age group

24. Distribution of study population by gender

25. Occurrence of gram negative and gram positive bacteria in single and mixed infection
Number
Percentage
Single infection 87
73.1%
Mixed infection 32
26.9%
Occurrence of gram negative and gram positive bacteria in single infection
Single infection
Number
Percentage
Gram negative bacteria 51
58.6%
Gram positive bacteria 36
41.4%
Occurrence of gram negative and gram positive bacteria in mixed infection
Mixed infection
Number
Percentage
Mixed growth of Gram negative bacteria 19
59.4%
Mixed growth of Gram positive bacteria 1
3.1%
Gram negative bacteria + Gram positive bacteria 12
37.5%

26. The most commonly isolated gram negative bacteria (n=103)
Pseudomonas aeruginosa (34.95%)
Enterobacter cloacae (14.56%)
Escherichia coli and Klebsiella pneumoniae (9.71% each)
Proteus mirabilis (8.74%)
Acinetobacter baumannii (5.83%)
Citrobacter koseri (2.91%)
Proteus valgaris, Citrobacter freundi and Serratia rubidiae (1.94% each)
Pseudomonas pudita, Enterobacter sakazakii, Escherichia hermanii, Klebsiella oxytoca, Citrobacter amalonaticu, Morganella morgani, Aeromonas hydrophila and Raoutella terrigena (0.97% for each isolate).

27. Type of ESBL-producing strains among Gram-negative bacteria isolated from orthopedic wound infection (n=13)

28. Antibiotic susceptibility pattern of Gram negative bacteria isolated in orthopedic wound infection
With regard to Pseudomonas species,
imipenem (78.38%)
piperacillin-tazobactam (67.57%)
ceftazidime (56.76%)
amikacin (43.24%)
cefepime (40.54%)
meropenem and tobramycin (37.84% each)
levofloxacin (35.14%)
ciprofloxacin (32.43%)
aztreonam (29.73%)
gentamicin (18.92%)

29. The isolated Acinetobacter baumannii were
50% sensitive to ampicillin/sulbactam and cefepime followed by
Ceftazadime, ciprofloxacin, levofloxacin, meropenem, gentamycin, tobramycin, amikacin, piperacillin/tazobactam, doxycycline and cotrimoxazole which were 33.33% sensitive
All of them were resistant to imipenem, cefotaxime and ceftriaxone.

30. For Enterobacteriaceae,
amikacin (81.66%)
cefepime (78.33%)
imipenem (76.67%)
ertapenam and piperacillin/tazobactam (73.33% each)
chloramphenicol (65%)
ceftazadime (60%)
aztreonam (56.67%)
cefoxitin (51.67%)

31. Out of 13 isolates of ESBL producing strains,
amikacin (84.62%)
imipenem (76.92%)
entrapenum (69.23%)
piperracillin/tazobactum and chloramphenicol (53.85%)
cefepime isolates (46.15%)
cefoxitin and meropenem, (46.2%)
aztreonam and tetracycline(30.8%)
gentamicin (23.1%)
norfloxacin (15.4%)
amoxicillin-clavulaniac acid and levofloxacillin (7.7%)

32. Antibiotic sensitivity pattern of ESBL producing bacteria isolates from orthopedic wound infection

33. Discussion

34. Occurrence of orthopedic wound infection
In the present study, out of 151 cases, bacterial pathogens were isolated in 119 cases (78.8%)
It was comparable with
study of May-Thu-Lwin (2016) in that bacterial pathogens were isolated in 60/115 (52.17%)

35. Occurrence of Gram negative bacteria in orthopedic wound infection
In the present study, out of 152 pathogenic bacteria, 103 isolates (67.76%) were gram negative bacteria
Meji-Soe-Aung (2003) revealed 62.07% (36/58) were gram negative bacteria
Aye-Mya-Thu (2010), 57.58% were gram negative bacteria and 42.62% were gram positive bacteria
Moe-Myint-Hein (2014) studied that the isolation rate of gram negative bacteria were 83.67% which were similar with the present study

36. In India, Jyoti et al., (2017), gram negative bacteria were isolated as 65%
In Indonesia done by Wardhana et al., (2017) stated that gram negative bacteria were isolated as 76%
In Malaysia, Leong et al.,(2017) revealed that of the pathogen isolated, 57% were gram negative bacilli

37. The reason for predominant of gram negative infection may be due to
prolong hospital stay
prophylaxis use of broad spectrum antibiotics,

38. Type of Gram negative bacteria in orthopedic wound infection
Pseudomonas aeruginosa (35%, 36/104)
Enterobacter cloacae (14%, 15/104)
Escherichia coli and Klebsiella pneumoniae (10%, 10/104 each)
Proteus mirabilis (9%, 9/104)

39. Htun-Oo-Saw (2005) -- Pseudomonas species (40%), Escherichia coli (12%) and Proteus species (7%)
Aye-Mya-Thu (2010) -- Pseudomonas species (31%), Escherichia coli (10%), Proteus species, Citrobacter species, Klebsiella species (5%)
Moe-Myint-Hein (2014) -- Pseudomonas species (22/50, 44%), Proteus species (6/50, 12%), Acinetobacter species (5/50, 10%), Escherichia coli (4/50, 8%)
May-Thu-Lwin (2016)-- Pseudomonas aeruginosa (27%), Escherichia coli (21%), Acinetobacter baumannii (12%), Klebsiella pneumoniae (11%), Enterobacter cloacae (8%),

40. In Vietnem, Le et al., (2006) -- Pseudomonas aeruginosa (30%) and Escherichia coli (10%)
In Cambodia, Hout et al., (2015) -- Esch. coli (20 %); Pseudomonas aeruginosa (14 %), Enterobacter cloacae (7 %,), Proteus mirabilis (7 %), Klebsiella pneumoniae (6 %)
In India, Shamanna et al. (2017) -- Pseudomonas aeruginosa constituted 36(19%), Enterobacter species 24(13%), Escherichia coli 14 (7%), Klebsiella Species 10(5%),
In Nigeria done by Idowu et al., (2011) -- Pseudomonas aeruginosa (18.62%) followed by Klebsiella spp.(17.64%) and Escherichia coli (14.7%).

41. The reason why Pseudomonas aeruginosa was the most isolated pathogen because it is a common cause of nosocomial infection as well an opportunistic pathogen
In the hospital, careful attention to routine infection control practices guided by
infection control committee
using antimicrobial agents according to policy

42. Occurrence of ESBL producing Gram negative bacteria
The incidence rate of ESBL producing gram negative bacteria in the present study were 12.5% (13/104)
Out of 13 isolates of ESBL-producing gram negative bacteria,
Klebsiella pneumonia was predominant (30.77%).
Enterobacter cloacae were 23.08%
Escherichia coli and Proteus miralbilis were 15.38% (2/13) for each isolate
Escherichia hermanii and Serratia rubidiae was 7.69% (1/13) for each isolate.

43. In Malaysia, 47 (10.6%) of ESBL-producing Klebsiella pneumoniae cultured from respiratory specimens(Loh et al., 2007)
In Bangladesh, Yasmin et al., (2015) studied that from 84 wound swabs, the most common ESBL producing bacteria were Klebsiella species (88.9%, 8/9) followed by Proteus species (78.3%, 18/23), and Escherichia coli (61.5%, 24/39).

44. The study by Sapai-Myint (2013), 16/235 isolates (6
The study by Sapai-Myint (2013), 16/235 isolates (6.8%) were ESBL producers which were Escherichia coli (75%) and Klebsiella pneumonia (25%).
Khin-Khin-Wai (2017), among 13 isolates (59.1%) of ESBL-producing Enterobacteriaceae, Escherichia coli was predominant (69.2%, 9/13), Enterobacter cloacae (15.4%, 2/13) and Klebsiella pneumoniae (15.4%, 2/13).

45. According to data from Myanmar, the prevalence of ESBL producing strains was varied but Esch. coli and Klebsiella spp. were stated as major ESBL producers
To reduce the risk of ESBL infection, standard infection control precautions guided by infection control committee must be used by all staff in all hospital settings
Moreover, avoiding indiscriminable usage of broad spectrum antibiotic should be necessary to reduce the occurrence of ESBL infection.

46. Antimicrobial Susceptibility Pattern of ESBL producing gram negative bacteria
Regarding ESBL producing gram negative bacteria in this study, the most sensitive drug were amikacin (84.62%), imipenem (76.92%), ertapenem(69.23%) and piperracillin/tazobactum and chloramphenicol (53.85% each)
Similarly, In Turkey Görmeli et al., (2015) stated that most effective antimicrobial drug for by ESBL producing strains were imipenem and amikacin

47. Khin-Khin-Wai (2017) studied that, among the isolates of, the most sensitive drug for ESBL producing Enterobacteriaceae was amikacin (76.9%) followed by cefoperazone-sulbactam (53.8%), piperacillin-tazobactam (30.8%),
Khin-Thawtar-Shein (2012) showing that sensitivity to imipenem was 96.7% of ESBL producing isolates and
Kaung-Htet (2014) finding that sensitivity to imipenem was 100%.

48. Antibiotic selection for infections with ESBL producing pathogens is still a clinical challenge
Inappropriate use antibiotic can result in increase resistant rate of these agents
Therefore, antimicrobial resistant rate among gram negative bacteria infection can be reduced by giving proper antibiotic treatment according to the guidline.

49. Conclusion

50. Although the occurrence of ESBL producing isolates in Myanmar is still not well defined,
laboratory-based surveillance should be continue yearly not only in tertiary centre but also in other district hospitals
All the results must be informed to clinicians regularly in order to evaluate the treatment guidelines of antimicrobial usage
In addition, it is important to control the nosocomial spread of drug resistant organism by organizing the infection control committee in every hospital

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