Open Access

Predominance of multi-resistant gram-negative bacteria colonizing chronic lower limb ulcers (CLLUs) at Bugando Medical Center

  • Nyambura Moremi1,
  • Martha F Mushi1Email author,
  • Mbunda Fidelis2,
  • Phillipo Chalya2,
  • Mariam Mirambo1 and
  • Stephen E Mshana1
Contributed equally
BMC Research Notes20147:211

DOI: 10.1186/1756-0500-7-211

Received: 18 September 2013

Accepted: 1 April 2014

Published: 4 April 2014

Abstract

Background

Infections, trauma, malignances and poorly controlled diabetes are common causes of chronic lower limb ulcerations in developing countries. Infected wound with multi-drug resistant bacteria usually are associated with increased morbidity and mortality. We report the distribution of bacteria pathogens colonizing the chronic lower limb ulcers and their drug susceptibility pattern from Bugando Medical Centre (BMC) a tertiary hospital in Tanzania.

Findings

Three hundred non-repetitive wound swabs were aseptically collected from 300 patients with chronic lower limb ulcers using sterile swabs and processed following standard operative procedures. Isolates were identified using in house biochemical testing and in case of ambiguous confirmation was done using API 20NE and API 20E. Susceptibility was determined using disc diffusion test following clinical laboratory standard Institute guidelines (CLSI). Of 300 swabs from patients with chronic lower limbs ulcers, 201 (67.7%) had positive aerobic culture within 48 hours of incubation. Of 201 isolates, 180(89.6%) were gram-negative bacteria. Out of 180 gram negative bacteria, resistance was detected for ampicillin (95%, n = 171), amoxicillin/clavulanate (83.9%, n = 151), trimethoprim-sulphamethoxazole (78.9%, n = 142), ceftriaxone (46.7%, n = 84), ceftazidime (45.6%, n = 82), gentamicin (39.4%, n = 71), ciprofloxacin (17.8%, n = 32) and meropenem 28(15.6%, n = 25). A total of 41 (35%) of enterobacteriaceae were found to be extended spectrum beta-lactamases (ESBL) producers while of 18 Staphylococcus aureus, 8(44.4%) were found to be methicillin resistant Staphylococcus aureus (MRSA).

Conclusion

There is high prevalence of ESBL and MRSA isolates in surgical wards at BMC. We recommend infection control and antibiotic stewardship programs in these wards to minimize spread of multi-resistant organisms.

Keywords

Chronic lower limb ulcers Multi-drug resistant Gram negative enteric

Findings

Background

In developing countries infections, trauma, malignances and poorly controlled diabetes are the most common causes of chronic lower limb ulcerations[1, 2]. An infected wound complicates the postoperative course and results in prolonged hospital stay and delayed recovery[3]. Prolonged hospital stay usually exposes the patient to health care associated infections (HCAs)[4], and more risk to infection due multi-drug resistant bacteria like extended spectrum beta lactamase (ESBL) producers and methicillin resistant Staphylococcus aureus (MRSA)[58]. In Tanzania about 50% of Klebsiella pneumonia and 25 -45% of Escherichia coli isolated from HCAIs are reported to be ESBL producers[911]. Also in Tanzania S. aureus has been reported to be the commonest cause of surgical site infections of which 18.8% are reported to be MRSA[3].

Clinical experience and outcome of patients regarding chronic lower limb ulcers in this centre has been described in previous publication[12]. This article is building from the same study but focusing on distribution of bacteria pathogens, susceptibility pattern of gram negative and gram positive isolates from chronic lower limb ulcers. In addition more information regarding the susceptibility pattern that predicts ESBL phenotype is presented.

The predominance of multi-drug resistant gram negative bacteria colonizing and infecting lower limbs necessitates the scaling up of infection control practices and the introduction of antibiotic stewardship in surgical wards in developing countries.

Methods

Study population

A cross sectional study involving all patients with chronic lower limbs ulcers was conducted between November 2011 and February 2012 in surgical wards of Bugando Medical Centre (BMC), a 1000 bed capacity tertiary hospital in the northwestern part of Tanzania as previously described[12].

Laboratory procedures

A total of 300 non-repetitive wound swabs were collected from 300 patients with chronic lower limb ulcers using sterile swabs. All swabs were processed as previously described[13]. Briefly specimens were inoculated on MacConkey agar and 7% sheep blood agar (HIMEDIA, INDIA) and incubated at 35°C aerobically for 24-48 hrs. Identification of bacteria was done using colonies characteristics, hemolysis on blood agar, lactose fermentation on differential media and in house biochemical tests using colonies from pure cultures; in case of ambiguous results confirmation was done using API 20NE and API 20E[11].

Antibacterial susceptibility testing

Susceptibility testing was performed by Kirby-Bauer technique. The 0.5 McFarland of test organism was uniformly seeded over the Mueller-Hinton agar surface and incubated at 35°C for 24 hours. Interpretation was made as per CLSI[14]. Antibiotics discs tested for gram positive bacteria included ampicillin (10 μg), cefoxitin (30 μg), trimethoprim-sulphamethoxazole (1.25/23.75 μg), gentamicin (10 μg), erythromycin (15 μg), clindamycin (2 μg) and vancomycin (30 μg). For gram negative bacteria, disc tested include ceftazidime (30 μg), amoxicillin/clavulanate (20/10 μg), ceftriaxone (30 μg), gentamicin (10 μg), trimethoprim-sulphamethoxazole (1.25/23.75 μg), ciprofloxacin (5 μg), meropenem (10 μg) and ampicillin (10 μg). ESBL producers were detected using disk approximation method as described previously[15, 16] while MRSA were detected using cefoxitin disc 30 μg, all isolates resistant to 30 μg cefoxitin based on CLSI were considered as MRSA[17]. In this study all isolates resistant to at least three of the following antimicrobials: ampicillin, amoxicillin/clavulanate, ceftazidime, ciprofloxacin, gentamicin, and/or trimethoprim-sulphamethoxazole (SXT) were considered as multi-drug resistant bacteria[18].

The study was approved by Bugando Medical Centre/Catholic University of Health and Allied Sciences ethics committee and the informed consent were obtained from all patients.

Results

A total of 300 patients with CLLUs were included in the study. Traumatic ulcers were the most frequent type of ulcer accounting for 181 (60.3%) of patients followed by infective ulcers 43 (14.3%), metabolic ulcers 35 (11.7%), neoplastic/malignant ulcers 20 (6.7%), vascular ulcers 11 (3.7%), neuropathic ulcers 8 (2.7%) and ulcerating skin lesions e.g. pyogenic granulomatous 2 (0.7%). Out of 300, 201 (67%) had positive aerobic culture within 48 hours of incubation. Of 201 positive cultures 180(89.6%) were identified as gram negative bacteria and 21(15.9%) as gram positive bacteria (Staphylococcus aureus 18, Enterococcus spp 3). Bacteria species isolated were; Pseudomonas spp. 54 (26.9%), Proteus spp. 45 (22.4%), Klebsiella pneumoniae 33 (16.4%), E. coli 26 (12.9%), Staphylococcus aureus 18 (9%), Acinetobacter spp. 9 (4.5%), Serratia spp. 7 (3.5%), Enterobacter spp. 6 (3%) and Enterococcus spp 3 (1.5%).

Of 180 gram negative bacteria, resistance was detected for ampicillin (95%, n = 171), amoxicillin/clavulanate (83.9%, n = 151), trimethoprim-sulphamethoxazole (78.9%, n = 142), ceftriaxone (46.7%, n = 84), ceftazidime (45.6%, n = 82), gentamicin (39.4%, n = 71), ciprofloxacin (17.8%, n = 32) and meropenem 28 (15.6%, n = 25) Table 1. A total of 41 (35%) of enterobacteriaceae were found to be ESBL producers, with specific ESBL rate for Escherichia coli and Klebsiella spp being 50% and 53% respectively. The resistance to trimethoprim-sulphamethoxazole, gentamicin and ciprofloxacin was found to predict ESBL phenotype (Table 2). Staphylococcus aureus were resistant to penicillin (79.3%), amoxicillin/clavulanate (60%), gentamicin (8.3%), trimethoprim-sulphamethoxazole (60%), clindamycin (20.7%), erythromycin (13.8%) and 8 (44.4%) were found to be MRSA.
Table 1

Resistance pattern of 180 gram negative isolates to various antibiotics in percentage

Bacteria isolates

N

AMP

CAZ

AMC

CRO

GM

SXT

CIP

MEM

E.coli

26

100

50.0

88.5

50.0

30.8

76.9

19.2

19.2

Klebsiella spp.

33

100

59.4

93.9

59.3

54.5

78.8

9.1

6.06

Pseudomonas spp.

54

100

55.1

92.6

55.1

44.4

96.3

20.4

25.9

Proteus spp.

45

88.9

20.0

71.1

20.0

22.2

57.8

17.8

2.2

*Other gm–ve spp

22

80.0

45.9

68.0

54.5

52.0

84.0

24.0

28.0

Total

180

95

45.6

83.9

46.7

39.4

78.9

17.8

15.6

Keys: AMP = Ampicillin, CAZ = Ceftazidime, AMC = Augmentin, CRO = Ceftriaxone GM = Gentamicin, SXT = trimethoprim-sulphamethoxazole, CIP = Ciprofloxacin, MEM = Meropenem. * Acinetobacter spp., Serratia spp. and Enterobacter spp.

Table 2

Antimicrobial resistance pattern that predict ESBL phenotype

Predictive drug resistance phenotype

ESBL n (%)

OR (95% CI)

p- value

Yes

No

  

AAGCS

    

  Yes

12 (44.4)

15 (55.6)

3.50 (1.5-8.3)

0.004

  No

29 (18.6)

127 (81.4)

1

 

AAG

    

  Yes

30 (44.8)

37 (55.2)

7.74 (3.5-17.0)

<0.001

  No

11(9.48)

105 (90.5)

1

 

AAC

    

  Yes

14 (43.8)

18 (56.3)

3.57 (1.6-8.1)

0.002

  No

27 (17.9)

124 (82.1)

1

 

GCS

    

  Yes

12 (42.9)

16 (57.1)

3.26 (1.4-7.6)

0.006

  No

29 (18.7)

126 (81.3)

1

 

GC

    

  Yes

12 (42.9)

16 (57.1)

3.26 (1.4-7.6)

0.006

  No

29 (18.7)

126 (81.3)

1

 

GS

    

  Yes

33 (45.8)

39 (54.2)

10.89 (4.6-25.6)

<0.001

  No

8 (7.2)

103 (92.8)

1

 

Note: AAGCS = A- Ampicillin, A- Amoxicillin/Clavulanate, G- Gentamicin, C-Ciprofloxacin, and S- trimethoprim-sulphamethoxazole, AAG = A- Ampicillin, A- Augmentin, G- Gentamicin, AAC = A- Ampicillin, A- Augmentin, C- Ciprofloxacin, GCS = G- Gentamicin, C- Ciprofloxacin, S- trimethoprim-sulphamethoxazole, GC = G- Gentamicin, C- Ciprofloxacin, GS = G- Gentamicin, S- trimethoprim-sulphamethoxazole.

Discussion

The microbiological profile of chronic ulcers of the lower limbs is very important in the provision of appropriate management of ulcers as well as institution-specific antibiotic policy in the surgical wards[19]. As reported previously[3], Pseudomonas aeruginosa was the most frequent gram negative bacteria isolated while Staphylococcus aureus was the commonest gram positive bacteria. Most of these isolates were multiply–resistant to commonly used antibiotics. This is due to the fact that most of these patients were hospitalized for more than 72 hrs signifying health care associated infections[3].

Compared to previous data in the surgical wards[3] the ESBL rates among Klebsiella spp and E. coli colonizing CLLU were lower than those involved in surgical site infections. Also this study confirmed the previous observation[10, 11] that the resistance to trimethoprim-sulphamethoxazole, gentamicin, and ciprofloxacin is a predictor of ESBL phenotype. Most of ESBL producers in this study are multi-drug resistance, due to the fact that most of ESBL conjugative plasmids observed previously in this settings[20, 21] carry resistance markers for other antibiotic classes such as tetracycline, gentamicin and trimethoprim-sulphamethoxazole. Similar to other studies[11, 22], majority of gram negative and gram positive isolates were sensitive to meropenem and vancomycin respectively. Increased trend was observed for MRSA at BMC whereby in 2009 and 2011 about 16.3% and 18.8% of S. aureus were found to be MRSA respectively[3, 17] while in the current study about 44% of Staphylococcus aureus were found to be MRSA.

Despite the importance of these data some limitations were failure to perform; anaerobic culture, molecular characterization and PCR confirmation of ESBL and MRSA phenotype.

Conclusion

High prevalence of ESBL and MRSA isolates were observed in surgical wards at BMC. We highly recommend Infection control and antibiotic stewardship programs in these wards to minimize spread of multi-resistant organisms. Coordinated surveillance of multi drug resistant isolates in Tanzania and other developing countries is highly needed so that this worldwide public health problem is controlled.

Notes

Declarations

Acknowledgements

We would like to acknowledge the assistance and guidance provided by all staff members of the department of Surgery, Bugando Medical Center and Microbiology/Immunology, Catholic University of Health and Allied Sciences, Bugando. This study was supported by reagents to SEM from SACIDS.

Authors’ Affiliations

(1)
Department of Microbiology/Immunology, Catholic University of Health and Allied Sciences
(2)
Department of Surgery, Catholic University of Health and Allied Sciences

References

  1. Margolis D, Bilker W, Santanna J, Baumgarten M: Venous leg ulcer: incidence and prevalence in the elderly. J Am Acad Dermatol. 2002, 46: 381-386. 10.1067/mjd.2002.121739.PubMedView ArticleGoogle Scholar
  2. Liedberg E, Persson B: Increased incidence of lower limb amputation for arterial occlusive disease. Acta Orthop Scand. 1983, 54: 230-234. 10.3109/17453678308996562.PubMedView ArticleGoogle Scholar
  3. Mawalla B, Mshana SE, Chalya PL, Imirzalioglu C, Mahalu W: Predictors of surgical site infections among patients undergoing major surgery at Bugando Medical Centre in Northwestern Tanzania. BMC Surg. 2011, 11 (1): 21-10.1186/1471-2482-11-21.PubMedPubMed CentralView ArticleGoogle Scholar
  4. Green JW, Wenzel RP: Postoperative wound infection: a controlled study of the increased duration of hospital stay and direct cost of hospitalization. Ann Surg. 1977, 185 (3): 264-268. 10.1097/00000658-197703000-00002.PubMedPubMed CentralView ArticleGoogle Scholar
  5. Knothe H, Shah P, Krcmery V, Antal M, Mitsuhashi S: Transferable resistance to cefotaxime, cefoxitin, cefamandole and cefuroxime in clinical isolates of Klebsiella pneumoniae and Serratia arcescens. Infection Nov-Dec. 1983, 11 (6): 315-317.View ArticleGoogle Scholar
  6. Noyal MJC, Menezes GA, Harish BN, Sujatha S, Parija SC: Simple screening tests for detection of carbapenemases in clinical isolates of nonfermentative Gram-negative bacteria. Indian J Med. 2009, 129: 707-712.Google Scholar
  7. Moquet O, Bouchiat C, Kinana A, Seck A, Arouna O, Bercion R, Breurec S, Garin B: Class D OXA-48 carbapenemase in multidrug-resistant enterobacteria, Senegal. Emerg Infect Dis. 2011, 17: 143-144. 10.3201/eid1701.100244.PubMedPubMed CentralView ArticleGoogle Scholar
  8. Alebachew T, Yismaw G, Derabe A, Sisay Z: Staphylococcus aureus burn wound infection among patients attending Yeketit 12 hospital burn unit, Addis Ababa, Ethiopia. Ethiop J Health Sci. 2012, 22: 3-Google Scholar
  9. Kayange N, Kamugisha E, Jeremiah S, Mwizamholya DL, Mshana SE: Predictors of positive blood culture and deaths among neonates with suspected neonatal sepsis in a tertiary hospital, Mwanza- Tanzania. BMC Pediatr. 2010, 10: 39-10.1186/1471-2431-10-39.PubMedPubMed CentralView ArticleGoogle Scholar
  10. Moyo J, Aboud S, Kasubi M, Lyamuya EF, Maselle SY: Antimicrobial resistance among producers and non-producers of extended spectrum betalactamases in urinary isolates at a tertiary Hospital in Tanzania. BMC Research Notes. 2010, 3: 348-10.1186/1756-0500-3-348.PubMedPubMed CentralView ArticleGoogle Scholar
  11. Mshana SE, Kamugisha E, Mirambo M, Chakraborty T, Lyamuya E: Prevalence of multiresistant Gram-negative organisms in a tertiary hospital in Mwanza, Tanzania. BMC Research Notes. 2009, 2: 49-10.1186/1756-0500-2-49.PubMedPubMed CentralView ArticleGoogle Scholar
  12. Mbunda F, Mchembe MD, Chalya PL, Rambau P, Mshana SE, Kidenya BR, Gilyoma JM: Experiences with surgical treatment of chronic lower limb ulcers at a tertiary hospital in northwestern Tanzania: a prospective review of 300 cases. BMC Dermatol. 2012, 12 (1): 17-10.1186/1471-5945-12-17.PubMedPubMed CentralView ArticleGoogle Scholar
  13. Cheesbough M: District Laboratory Practice in Tropical Countries Part 2 second edition, vol. part 2. 2006, Cambrige University PressView ArticleGoogle Scholar
  14. Clinical and Laboratory Standards institute: Performance Standard for Antimicrobial Susceptibility Testing 20th Informational Suplliment vol. 30. 2010Google Scholar
  15. M'Zali F, Chanawong A, Kerr K, Birkenhead D, Hawley P: Detection of extendedspectrum beta-lactamases in members of the family Enterobacteriaceae: comparison of the Mast DD test, the double disc and the E-test ESBL. Antimicrob Agent Chemother. 2000, 45: 881-885. 10.1093/jac/45.6.881.View ArticleGoogle Scholar
  16. Gheldre Y, Avesami V, Berhin C, Delmee M, Glupenzynskii Y: Evaluation of OXOID combinations disc for detection of extended β-Lactamases. J Antimicrob Chemother. 2003, 52: 591-597. 10.1093/jac/dkg415.PubMedView ArticleGoogle Scholar
  17. Mshana S, Kamugisha E, Mirambo M, Chalya P, Rambau P, Mahalu W, Lyamuya E: Prevalence of clindamycin inducible resistance among methicillin-resistant Staphylococcus aureus at Bugando Medical Centre, Mwanza, Tanzania. Tanzania J Health Res. 2009, 11: 2-Google Scholar
  18. Mushi MF, Mshana SE, Imirzalioglu C, Bwanga F: Carbapenemase genes among multidrug resistant gram negative clinical isolates from a tertiary hospital in Mwanza, Tanzania. BioMed Res Int. 2014, 2014:Google Scholar
  19. Lim TS, Bibombe PM, Ronan M, Kishore S, Manzoor A, Donna A: Microbiological profile of chronic ulcers of the lower limb: A prospective observational study cohort study. ANZ J Surg. 2006, 76 (8): 688-692. 10.1111/j.1445-2197.2006.03832.x.PubMedView ArticleGoogle Scholar
  20. Mshana S, Imirzalioglu C, Hain T, Domann E, Lyamuya E, Chakraborty T: Multiple ST clonal complexes, with a predominance of ST131, of Escherichia coli harbouring blaCTX‒M‒15 in a tertiary hospital in Tanzania. Clin Micro Infect. 2011, 17 (8): 1279-1282. 10.1111/j.1469-0691.2011.03518.x.View ArticleGoogle Scholar
  21. Mshana SE, Hain T, Domann E, Lyamuya EF, Chakraborty T, Imirzalioglu C: Predominance of Klebsiella pneumoniae ST14 carrying CTX-M-15 causing neonatal sepsis in Tanzania. BMC Infect Dis. 2013, 13 (1): 466-10.1186/1471-2334-13-466.PubMedPubMed CentralView ArticleGoogle Scholar
  22. Seni J, Najjuka CF, Kateete DP, Makobore P, Joloba ML, Kajumbula H, Kapesa A, Bwanga F: Antimicrobial resistance in hospitalized surgical patients: a silently emerging public health concern in Uganda. BMC Research Notes. 2013, 6 (1): 298-10.1186/1756-0500-6-298.PubMedPubMed CentralView ArticleGoogle Scholar

Copyright

© Moremi et al.; licensee BioMed Central Ltd. 2014

This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly credited. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.

Advertisement