The Global Journal of Pharmaceutical Research Vol. 1(4), pp. 708-714, 15 Sep, 2012; www.tgjpr.com

Odumosu et al. 708

Resistance to expanded-spectrum cephalosporins in Pseudomonas aeruginosa

and other Gram-negative bacteria is mediated by extended-spectrum beta

lactamases (ESBLs). ESBL-producing bacteria have been responsible for clinical

failures and several outbreaks in hospitals when not accurately and promptly

detected. The purpose of this study was to phenotypically detect the production

of ESBLs among 54 multidrug resistant clinical isolates of P. aeruginosa obtained

from various specimens in 5 hospitals in Southwest Nigeria. The presence of

ESBL was determined by double disk synergy test (DDST) using

amoxicillin/clavulanate as the ESBL inhibitor. Of the 54 isolates, 5 (9.3%)

displayed a clear production of ESBL by showing a synergy towards the

clavulanic acid. A low rate of detection of ESBL by DDST despite a high

cephalosporin resistance rate of 53.7% observed in this study suggests the

presence of other β-lactamases not easily detected phenotypically by DDST.

Consequently, a combination of phenotypic and molecular detection methods

would be essential for a reliable epidemiological investigation of the diverse

groups of ESBLs produced by Pseudomonas aeruginosa and other Gram-

negative bacteria. Routine surveillance of antimicrobial resistance and robust

detection of ESBL among strains of P. aeruginosa is clinically and

epidemiologically important to forestall rapid spread and transfer of resistance

and ESBL genes among other nosocomial pathogens.

Key words: ESBL, Pseudomonas aeruginosa, multidrug, resistance, DDST.

INTRODUCTION

Pseudomonas aeruginosa is commonly

associated with nosocomial infections

such as pneumonia, urinary tract

infections and bacteremia1 Expanded-

spectrum cephalosporins such as

cefotaxime and ceftazidime are good

antipseudomonas drugs commonly

employed in the treatment of infections

caused by P. aeruginosa strains but

resistance to one or more of these drugs

has been reported2 due to the production

of extended-spectrum beta-lactamases

(ESBLs) by these strains. ESBLs are

mostly plasmid-borne enzymes produced

FULL LENGTH ORIGINAL ARTICLE ISSN 2277- 5439

Phenotypic detection of extended-spectrum beta-lactamase producing Pseudomonas aeruginosa from Hospitals in Southwest

Nigeria

Odumosu Bamidele .T,1 Adeniyi Bola A.,

1* Soge Olusegun O.,

2 Dada-Adegbola

Hannah O.

3

1Department of Pharmaceutical Microbiology, University of Ibadan, Ibadan, Nigeria,

2 Department of Global Health, University of Washington, Seattle, Washington, USA.

3Department of Medical Microbiology, University of Ibadan, Ibadan, Nigeria.

Corresponding Address: baadeniyi@yahoo.co.uk

Accepted 1

st Sep, 2012

The Global Journal of Pharmaceutical Research Vol. 1(4), pp. 708-714, 15 Sep, 2012; www.tgjpr.com

Odumosu et al. 709

in bacteria that permits hydrolysis of all

penicillins, first, second and third

generation cephalosporins and

aztreonam (but not cephamycins or

carbapenems) and are inhibited by

clavulanate3. The production of ESBLs

in bacteria, conferring resistance to wide

array of antibiotics were initially

associated with members of

Enterobactericeace such as K.

pneumoniae and E. coli, but have

spread to other Gram-negative bacteria

of other genera such as P. aeruginosa.

Incidence of P. aeruginosa strains

producing multiple ESBLs is increasing

around the world including Africa [4 – 7].

High rates (14 – 45%) of ESBL

production among clinical strains of P.

aeruginosa have been previously

reported in Nigeria 8 – 11

Double disk synergy test (DDST) method

using a beta-lactamase inhibitor such as

clavulanic acid was first designed and

most widely employed for the detection

of ESBL because it is cheap and easy to

interpret.12 Other ESBL detection tests

that have been proposed include three-

dimensional test method (TDT),13 Etest

ESBL strips14, Vitek 2 ESBL test15–16 and

clinical microbiology screening and

confirmatory test method17. However,

DDST remains the most reliable method

of all, due to its reproducibility and ease

of interpretation.

Inappropriate approach or failure

to respond to medical complications

caused by multidrug resistant bacteria

often leads to outbreaks, due to the ease

of dissemination and transfer of ESBL

genes within the hospital environment by

Gram-negative bacteria. Outbreaks

caused by ESBL producing P.

aeruginosa and other Gram-negative

bacteria have been reported. 18 - 20

Frequent detection of ESBL producing

bacteria such as P. aeruginosa is

clinically and epidemiologically important;

and data on the current status of ESBL

among strains of P. aeruginosa will

enable effective empirical treatment of

infections caused by such strains. Our

aim was to investigate the prevalence of

ESBL producing P. aeruginosa isolates

from 5 hospitals in 3 Southwestern

States of Nigeria using the internationally

recognized DDST.

MATERIALS AND METHODS

Bacteria strains

Fifty-four non-duplicate clinical isolates of

P. aeruginosa were recovered over a

period of six months (March 2010 - Sept

2010) from variety of specimens,

including urine, pus, wound swab, ear

swab, blood and vaginal swab from five

different tertiary hospitals in Southwest

Nigeria.

Isolation and identification

All strains were previously isolated and

identified from various specimens as P.

aeruginosa at Medical Microbiology units

of tertiary hospitals in three Southwest

States of Nigeria. P. aeruginosa

identities were further confirmed by

standard biochemical methods as

previously described. 21

The Global Journal of Pharmaceutical Research Vol. 1(4), pp. 708-714, 15 Sep, 2012; www.tgjpr.com

Odumosu et al. 710

Antimicrobial susceptibility testing

Antimicrobial susceptibility testing of the

isolates against β-lactamase inhibitors

(piperacillin/tazobactam and

ticarcillin/clavulanate) 3rd generation

(cefotaxime, ceftriaxone, ceftazidime),

4th generation cephalosporin (cefepime)

monobactam (aztreonam) and

carbapenem (imipenem) were done as

previously reported 22. E. coli 25922 and

P. aeruginosa ATCC 27853 were used

as quality controls.

Double-disk synergy test

DDST for all the cephalosporin-resistant

and susceptible strains was performed

as a standard disk diffusion assay on

Mueller-Hinton agar as described by

Jarlier et al.12 with modifications. Disks

containing 30 μg of aztreonam,

ceftazidime, ceftriaxone, and cefotaxime,

were placed 20 mm and 15 mm apart

(centre to centre) consecutively from a

disk containing amoxicillin (20 μg) plus

clavulanic acid (10 μg) and incubated for

18 - 24 h at 37oC. Enhancement of the

inhibition zone towards the amoxicillin-

clavulanate disc, indicating synergy

between clavulanic acid and any one of

test antibiotics, was regarded as

presumptive ESBL production.1, 12

RESULTS

Twenty-nine (53.7%) of the 54 P.

aeruginosa were resistant to two or more

expanded-spectrum cephalosporins. The

highest rates of resistance were

observed for ceftriaxone (53.7%),

cefotaxime (51.9%) and aztreonam

(44.4%). The resistance rates of 16.7%

and 18.6% were observed for

ceftazidime and cefepime respectively.

Of the two β-lactamase inhibitors,

ticarcillin/clavulanate gave a higher

resistance rate of 87.0% compared to

piperacillin/tazobactam (38.9%). Most of

the isolates from this study were

susceptible to imipenem, with resistance

observed for only 7.5% isolates.

Interestingly, ESBL was detected in only

5 (9.3%) of 54 isolates investigated,

while 24 (44.4%) cephalosporin-resistant

isolates suspected to be ESBL-

producers showed no synergy with

clavulanic acid. The remaining 25

(46.3%) were cephalosporin-susceptible

and were also ESBL-negative by DDST.

ESBL was detected at the 15 mm

distance in 4 cephalosporin-resistant

isolates and at the 20 mm distance from

the β-lactamase inhibitor disk for P.

aeruginosa strain ODM 46 (Table 1).

Synergy was common at the ceftazidime

and cefotaxime disks towards the

amoxicillin/clavulanate among the 5

positive strains. All the 5 ESBL positive

strains were susceptible to imipenem.

DISCUSSION

P. aeruginosa demonstrates a variety of

enzymatic and mutational mechanisms

of bacterial resistance [23, 24] Often

times, these mechanisms are seen to

manifest simultaneously, thus conferring

combined resistance to many strains.24,25

Low prevalence of ESBL by DDST

among P. aeruginosa was observed in

this study. Only 5 (9.3%) isolates showed

synergy in the presence of the β-

lactamase inhibitor (clavulanic acid)

while the remaining ESBL suspected

The Global Journal of Pharmaceutical Research Vol. 1(4), pp. 708-714, 15 Sep, 2012; www.tgjpr.com

Odumosu et al. 711

strains, which were resistant to the ESBL

marker antibiotics remained negative

even at a reduced distance of 15 mm of

cephalosporin to the

amoxicillin/clavulanate disk. Similar

distance of 15 mm for DDST has been

previously shown to be most reliable for

detecting ESBL26. Previous studies in

Nigeria have documented higher

prevalence of ESBL in P. aeruginosa by

DDST method. For instance Aibinu et al.8

reported 45% detection of ESBL among

clinical strains of P. aeruginosa

investigated in 2 hospitals in Lagos while

Akinjogunola et al.9 reported 30%

detection among isolates obtained from

UTI in the South-South Nigeria. In

another study, Osazuwa et al10 reported

14% detection among P. aeruginosa

investigated along with other Gram-

negative bacteria isolated from HIV

infected patients in Benin Metropolis

while in a recent study by Okesola and

Oni11, 22.2% rate of detection was

reported from clinical isolates from

University College Hospital in Ibadan.

However, a lower rate of detection of

ESBL in this study compared to the

previous reports might be due to the

presence of other resistance genes

conferring multiple resistances to the

investigated strains. Similar low rates of

4.0% and 8.1% of ESBL detection with

DDST have been reported in Turkey and

Iran respectively. 27, 28

The antimicrobial susceptibility

results showed that the P. aeruginosa

from this study were resistant to 3rd

generation cephalosporins especially

ceftriaxone (53.7%) and cefotaxime

(51.9%), and were also resistant to

aztreonam (44.4%) suggesting the

presence of ESBL among the resistant

strains. Difficulty in the phenotypic

detection of ESBL by DDST in P.

aeruginosa has been previously

reported, been hazarded by the frequent

chromosomal β-lactam resistance

mechanisms such as the over-

expression of AmpC β-lactamase and/or

one of the several efflux pumps encoded

in its genome.29-31 AmpC β-lactamase

resist clavulanic acid hence prevents

synergy between β-lactam and clavulanic

acid. Resistance of the isolates in this

study against ticarcillin/clavulanate

(87.0%) and piperacillin/tazobactam

(38.9%) indicates the presence of AmpC

β-lactamase among these strains.

Additionally, P. aeruginosa strains in this

study have also been shown to be

resistant to other classes of antimicrobial

agents including fluoroquinolones and

aminoglycosides 22. This further suggests

the presence of other resistance

structures such as outer membrane

impermeability, efflux pumps and

integrons that are associated with

multidrug resistance and capable of

masking ESBL detection among the

suspected strains in this study.

Tzelepi et al.32 suggested the use

of cefepime to inhibit the activities of the

AmpC enzymes and efflux pumps in P.

aeruginosa thereby increasing the

chance of the detection of ESBL by

DDST. However, based on the

resistance data obtained in this study,

inclusion of cefepime may have shown

little or no effect especially among the

The Global Journal of Pharmaceutical Research Vol. 1(4), pp. 708-714, 15 Sep, 2012; www.tgjpr.com

Odumosu et al. 712

18.6% isolates that were resistant to

cefepime, suggesting interplay of

multiple resistance mechanisms among

the P. aeruginosa strains. The low

sensitivity of DDST for detecting ESBL

among the cephalosporin-resistant

isolates from this study could also be

indicative of the presence of different β-

lactamases, which are not easily

detected by conventional phenotypic

ESBL detection methods such as DDST.

Therefore, data on the presence of ESBL

among clinical P. aeruginosa obtained by

DDST could be insufficient to assess

prevalence of ESBLs. Previous studies

have documented tazobactam inhibitory

activity against ESBL and AmpC beta-

lactamase to be almost 10 fold greater

than clavulanic acid33,34. Tazobactam

could therefore be used as a beta-

lactamase inhibitor for DDST method

along with cefepime or cloxacillin,

especially for bacteria that co-produce

ESBL and AmpC beta-lactamases.

However, a combination of phenotypic

and molecular detection methods

remains the best reliable, robust

surveillance system for detecting the

diverse group members of the ESBLs.

This will improve the empirical treatment

and management of infections caused by

ESBL producing bacteria.

In conclusion, resistance to

expanded-spectrum cephalosporins

among clinically and epidemiologically

important Gram-negative bacteria

including P. aeruginosa should serve as

a warning signal to the presence of

ESBL; and the detection of such ESBL-

producing strains should necessitates

the implementation of isolation

procedures to prevent outbreaks arising

from cross-transmission to other

patients. Importantly, early and accurate

detection of ESBL-producing P.

aeruginosa and other Gram-negative

bacteria is crucial for effective treatment

and control of the rapid spread of

plasmid-encoded ESBL genes among

these pathogens. Further studies will be

carried out to characterize all the

cephalosporin-resistant isolates from this

study by molecular methods involving

polymerase chain reaction and

sequencing.

REFERENCES 1. Pagani L, Mantengoli E, Migliavacca R et al.

“Multifocal detection of multidrug-resistant Pseudomonas aeruginosa producing the PER-1 extended spectrum beta-lactamase in northern Italy”. J Clin Microbiol,; 39:1865–1870, 2004.

2. Tam V.H, Chang K.T, Abdelraouf K, Brioso C.G, Ameka M, McCaskey L.A, Weston J.S et al., 2010 “Prevalence, Resistance Mechanisms, and Susceptibility of Multidrug-Resistant Bloodstream Isolates of Pseudomonas aeruginosa Antimicrobial” Agents Chemotherapy 54: 1160–1164, 2010.

3. Paterson D. L, Bonomo R. A. “Extended-Spectrum β -Lactamases: a Clinical Update” Clin Microbiol Rev.18: 657–686 , 2005.

4. Jones R.N. “Resistance patterns among nosocomial pathogens: trends over the past few years”. Chest; 11: 397-404, 2001.

5. Weld H.G, Poirel L, Nordmann P. “Ambler class A extendedspectrum β-lactamase in Pseudomonas aeruginosa: novel development and clinical impact”. Antimicrob Agents Chemother; 47:2385-2392, 2003.

6. Poirel L, Weldhagen G.F, De Champs C, Nordmann P.A “Nosocomial outbreak of Pseudomonas aeruginosa isolates expressing the extended-spectrum beta-lactamase GES-2 in South Africa”. J Antimicrob Chemother. 49: 561-565, 2002.

7. Mansour W, Dahmen S, Poirel L, Charfi K, Bettaieb D, Boujaafar N, Bouallegue O.

The Global Journal of Pharmaceutical Research Vol. 1(4), pp. 708-714, 15 Sep, 2012; www.tgjpr.com

Odumosu et al. 713

“Emergence of SHV-2a extended-spectrum beta-lactamases in clinical isolates of Pseudomonas aeruginosa in a university hospital in Tunisia”. Microb Drug Resist.15: 295-301, 2009.

8. Aibinu I, Nwanneka T , Odugbemi T “ Occurrence of ESBL and MBL in Clinical Isolates of Pseudomonas aeruginosa From Lagos”, Nig J Ame Sc. 3:81-85, 2007.

9. Akinjogunla O. J, Odeyemi A. T, Olasehinde G. I. “Epidemiological Studies of Urinary Tract Infection (UTI) among Post-menopausal Women in Uyo Metropolis, South-South, Nigeria”. J Amer Sci. 6, 1674-1681, 2010

10. Osazuwa F, Osazuwa E.O Imade P. E. Dirisu J. O. Omoregie R Okuonghae P E Aberare L “Occurrence of extended spectrum beta-lactamase producing Gram negative bacteria in HIV AIDS infected patients with urinary and gastrointestinal tract infections in Benin metropolis”. RJPBCS 2, 230-234, 2011.

11. Okesola O.A, Oni A.A. “Occurrence of Extended-spectrum beta-lactamase producing Pseudomonas aeruginosa strains in South-West Nigeria”. Res J Med Sci 6: 93-96, 2012.

12. Jarlier, V, Nicolas M. H, Fournier G, Philippon A. 1988. “Extended broad-spectrum beta-lactamases conferring transferable resistance to newer beta-lactam agents in Enterobacteriaceae: hospital prevalence and susceptibility patterns”. Rev. Infect. Dis. 10:867–878, 1988.

13. Thomson, K. S., and Sanders C. C. “Detection of extended-spectrum β-lactamases in members of the family Enterobacteriaceae: Comparison of the double-disk and three-dimensional tests”. Antimicrob. Agents Chemother. 36:1877–1882, 1992

14. Vercauteren, E., P. Descheemaeker, M. Leven, C. C. Sanders, and H. Goossens.. “Comparison of screening methods for the detection of extended-spectrum β-lactamases and their prevalence among blood isolates of Escherichia coli and Klebsiella spp. in a Belgian teaching hospital” J. Clin. Microbiol. 35:2191–2197, 1997

15. Sanders, C. C, Barry A. L, Washington J. A., Shubert C., Moland E. S., Traczewski M. M., Knapp C., and Mulder R. “Detection of extended-spectrum β-lactamases producing members of the family Enterobacteriaceae

with the Vitek ESBL test”. J. Clin. Microbiol. 34:2997–3001, 1996.

16. Tenover, F. C, Mohammed J. M, Gorton T, and Dembek Z. F. “Detection and reporting of organisms producing extended-spectrum beta-lactamases: survey of laboratories in Connecticut”. J. Clin. Microbiol. 37:4065–4070, 1999.

17. Clinical and Laboratory Standards Institute (CLSI). “Performance Standards for Antimicrobial Susceptibility Testing; Twentieth Informational Supplement”. 2010: CLSI Document M100- S20, Wayne, PA: Clinical and Laboratory Standard Institute.

18. Karas J.A, Pillay D.G, Muckart D, Sturm A.W. “Treatment failure due to extended spectrum beta-lactamase”. J Antimicrob Chemother; 37:203-04, 1996.

19. Eveillard M, Biendo M, Canarelli B, Daoudi F, Laurans G, Rousseau F, Thomas D. “Spread of Enterobacteriaceae producing broad-spectrum beta-lactamase and the development of their incidence over a 16-month period in a university hospital center].” Pathol Biol; 49 :515-21, 2001

20. Buré A, Legrand P, Arlet G, Jarlier V, Paul G, Philippon A. “Dissemination in five French hospitals of Klebsiella pneumoniae serotype K25 harbouring a new transferable enzymatic resistance to third generation cephalosporins and aztreonam.” Eur J Clin Microbiol Infect Dis;7:780-2, 1988.

21. Cheesbrough M. “District laboratory practice in tropical countries Part 2”, Cambridge University Press. 2000.

22. Odumosu B.T, Adeniyi B.A, Dada-Adegbola H, Chandra R. “Multidrug resistant Pseudomonas aeruginosa from Southwest Nigeria hospitals” Int. J. Pharm. Sci. Rev. Res., 15: 11-15, 2012.

23. Bert F, Ould-Hocine Z, Juvin M, DuboisV, Loncle-Provot V, Lefranc V, Quentin C , Lambert N , and Arlet G.

“Evaluation of the Osiris Expert System for Identification of β-Lactam Phenotypes in Isolates of Pseudomonas aeruginosa” J Clin Microbiol. 41: 3712–3718, 2003

24. Pechere, J. C Kohler T. “Patterns and modes of β-lactam resistance in Pseudomonas aeruginosa”. Clin Microbiol Infect 5 :15–18, 1999.

25. McGowan J. E. “Resistance in non-fermenting Gram-negative bacteria: multidrug resistance to the maximum”. Am J Infect Control 34, 29–37, 2006.

The Global Journal of Pharmaceutical Research Vol. 1(4), pp. 708-714, 15 Sep, 2012; www.tgjpr.com

Odumosu et al. 714

26. John S, Balagurunathan R. “Metallo beta lactamase producing Pseudomonas aeruginosa and Acinetobacter baumannii” Indian J Med Microbiol. 29; 302-304, 2011

27. Gençer S, Öznur A.k, Benzonana N, Batırel A and Özer S. Susceptibility patterns and cross resistances of antibiotics against Pseudomonas aeruginosa in a teaching hospital of Turkey. Annals of Clin Microbiol Antimicrob, 1:1-4, 2002.

28. Tavajjohi Z, Rezvan Moniri Ahmad K “Detection and characterization of multidrug resistance and extended-spectrum-beta-lactamase-producing (ESBLS) Pseudomonas aeruginosa isolates in teaching hospital Zahra” African Journal of Microbiology Research. 5: 3223-3228, 2011

29. Vahaboglu H, Ozturk R , Akbal H, Saribas S, Tansel O, Coskunkan F. “Practical Approach for Detection and Identification of OXA-10-Derived ceftazidime-hydrolyzing extended-spectrum β-Lactamases” J Clin Microbiol 36 : 827–829, 1998.

30. Aubert D, Girlich D, Naas T. Nagarajan S, Nordmann P. “Functional and structural characterization of the genetic environment of an extended-spectrum beta-lactamase blaVEB gene from a Pseudomonas aeruginosa isolate obtained in India”.

Antimicrob. Agents Chemother. 48:3284-3290, 2004.

31. Juan C, Mulet X, Zamorano L, Albertí S, Pérez J.L. Oliver A. “Detection of the Novel Extended Spectrum β-lactamase (ESBL) OXA-143 from a Plasmid-Located Integron in Pseudomonas aeruginosa Clinical Isolates in Spain” Antimicrob. Agents Chemother doi:10.1128/AAC.00822-09. 2009.

32. Tzelepi E, Giakkoupi A, Sofianou D, Loukova V, Kemeroglou A, Tsakris A. “Detection of extended-spectrum β-lactamases in clinical isolates of Enterobacter cloacae and Enterobacter aerogenes”. J. Clin. Microbiol. 38:542-546, 2000.

33. Bush K, Macalintal C, Rasmussen B.A, Lee V.J and Yang Y. “Kinetic interaction of tazobactam with beta-lactamases from all major structural classes”. Antimicrob Agents Chemother. 37: 851-858, 1993

34. Phillippon A, Arlet G, Jacoby G.A. “Plasmid-determined AmpC type β-lactamases”. Antimicrob Agents Chemother; 46: 1-11, 2002.

Table 1: DDST diameter of the zones of inhibition of ESBL marker antibiotics and cephalosporin

resistance patterns

Strain Clinical source

ESBL status Zones of inhibition of ESBL marker antibiotics (mm)(CAZ, CTX, ATM)

Cephalosporin resistance patterns

ODM 5 Pus Positive 16 15 18 CAZ,CRO, CTX

ODM 8 Wound Positive 25 20 25 CRO, CTX

ODM 17 Urine Positive 20 15 22 CRO, CTX

ODM 42 Pus Positive 15 6 17 CAZ, CRO, CTX

*ODM 46 Urine Positive 18 23 20 CAZ, CRO, CTX, FEP ATM= aztreonam, CAZ= ceftazidime, CRO= ceftriaxone, CTX= cefotaxime, FEP= cefepime * ODM 46 was detected at the 20mm distance from the amoxicillin/clavualante disk