Antimicrobial Resistance and Prevalence of Extended Spectrum Beta-Lactamase Proteus spp. Strains from Southwestern Nigeria Hospitals. Alabi, O. S.1, Mendonça, N.2, Adeleke, O. E.1, and Da Silva, G. J.2
23rd ECCMID
1Department 2Center
P1306
[email protected]
of Pharmaceutical Microbiology, Faculty of Pharmacy, University of Ibadan, Ibadan, Nigeria.
of Pharmaceutical Studies and Laboratory of Microbiology, Faculty of Pharmacy, University of Coimbra, Coimbra, Portugal.
ABSTRACT
RESULTS
Objectives: The main goal of the study was to determine antibiotic susceptibility and the prevalence of extended-spectrum beta-lactamase (ESBL) producing Proteus spp. isolated from clinical samples from southwestern Nigeria hospitals. Methods: Proteus spp. isolates (n=120) were collected from diverse clinical specimens in hospitals from four states in the Southwest of Nigeria during 2011, and identified by Microbact 12/24E (Oxoid). Antibiotic susceptibility was done by disc diffusion method against 13 antibiotics, and MICs determined by microdilution for ESBL positive strains, as suggested by the double disk synergy test. ESBL encoding genes and the insertion gene ISEcp1 were screened by PCR for the most common beta-lactamases: TEM, SHV and CTX-M. Amplicons were sequenced and identified using BLAST software. Results: Of the 120 Proteus spp. (P. mirabilis, 110 and P. vulgaris 10), 45.8% and 54.2% are from male and female patients, respectively. The majority were isolated from wound swabs (40.8%), followed by urine (22.5%), ear swabs (23,3%), stool (3.3%), endo-cervical swab (2.5%), high vagina swab (1.7%), eye swab (1.7%), sputum (1.7%), and pus (0.8%). The antibiotic susceptibility test showed that 66.4% of the isolates were resistant to amoxicillin, 17.4% to amoxicillin/clavulanic acid, 31.5% to cefotaxime, 23.2% to ceftazidime, 14.1% to aztreonam, 8.3% to cefoxitin, 2.5% to imipenem, 24.9% to gentamicin, 4.2% to amikacin, 48.8% to nalidixic acid, 14.1% to ciprofloxacin, 72.2% to sulphamethoxazole/trimethoprim and 1.7% to tigecycline. The phenotypic ESBL detection suggested 24.2% ESBLs. All strains showed a bla TEM-like enzyme. The blaCTX-M-15 was identified in four P. mirabilis (3.3%) from wounds (one from a six year old child), that also produced TEM-1. No SHV beta-lactamases were found. Two of the blaCTX-M-15 genes were associated with the ISEcp1 element. Imipenem and amikacin were active against 75% of ESBL-producers; while tigecycline showed activity against 100%. They were resistant to ciprofloxacin and gentamicin. Conclusions: To our knowledge, this is the first study in Nigeria showing the presence of the CTX-M-15 in Proteus sp. mostly isolated from wounds. The study highlight the dissemination of this ESBL in Enterobacteriaceae other than Escherichia coli, and the high potential spread in Nigerian hospitals, demanding the use of more expensive antibiotics such as imipenem or tygecycline.
Antibiotic susceptibility patterns of of Proteus spp. clinical isolates 90
Percentage of sex distribution
80
ESBLs were produced by 24.2% of the isolates mostly by P. mirabilis from wound infections (44.8% of the ESBL-producers) and 54.2% were multidrug resistance.
% isolates
70
46%
54%
60 50
Resistant Intermediate Sensitive
40 30
10
Male
0
Female
CTX
CAZ
FOX
ATM
IPM
GN
AK
AML
AMC
NA
CIP
SXT
TGC
Antibiotics CTX, Cefotaxime; CAZ, Ceftazidime; FOX, Cefoxitin; ATM, Aztreonam; IPM, Imipenem; GN, Gentamicin; AK, Amikacin; AML, Amoxicillin; AMC, Amoxicillin/clavulanic acid; NA, Nalidixic acid; CIP, Ciprofloxacin; SXT, Sulphamethoxazole/Trimethoprim; TGC, Tigecycline.
% Distribution of ESBL-producers by sex
Proteus spp. have been implicated in several nosocomial outbreaks and community acquired infections (Samuel et al, 2010; Gebre-Sealsssie, 2007) in some parts of the world including Nigeria (Chikere et al., 2008). The most common infections caused by Proteus spp. are urinary tract infections (UTIs), pneumonia, septiceamia and wound infections (Okesola et al., 2010). Clinical isolates of Proteus spp. have been found to harbour various types of extended-spectrum beta-lactamase resistant encoding genes conferring resistance to narrow, broad and extended-spectrum antibiotics. Some of these genes so far isolated in Proteus spp. are blaTEM, blaSHV, blaCTX-M, blaOXA and blaGES (Song et al., 2011; Peng et al., 2011; Bonnet et al., 2002; Thierry et al., 2000). Mobile genetic elements like ISEcp1, IS26, integrons and transposons responsible for the capture and acquisition of resistant genes (Poirel et al., 2005) have also been reported in Proteus spp. In Nigeria, the knowledge on the prevalence of ESBLs in Proteus spp. is scarce. Therefore, this study aimed at determining the susceptibility patterns of clinical isolates of Proteus spp. from Southwest Nigeria against some antibiotics as well as the prevalence of ESBLs.
% Distribution of ESBL-producers by Region
70 60
CONCLUSIONS
Percentage (%)
Percentage (%)
60 50
40
30
50
This study confirms the presence of the 40
CTX-M-15 in Proteus mirabilis in 30
Nigeria,
solely isolated from wounds.
20
20
The study highlights the dissemination of
10
10
this ESBL in Enterobacteriaceae other than Escherichia coli, and the high potential
0
0
Male
One hundred and twenty clinical isolates of Proteus spp. were collected from diverse clinical specimens in five hospitals from four states in the Southwest of Nigeria in 2011, and characterized into species by Microbact 12E (Oxoid).
OYO
Female
Sex
OGUN
OSUN
LAGOS
Regions
spread in Nigerian hospitals, demanding the use of more expensive antibiotics such as
% Distribution of ESBL-producers by Pathological Sources
Antibiotic susceptibility tests Antibiotic susceptibility test was performed by disc diffusion method using 13 antibiotic.The minimum inhibitory concentrations (MICs) of two cephalosporins used in the ESBL screening (ceftazidime and cefotaxime) with and without clavulanic acid were determined by microdilution broth method according CLSI 2011 guidelines for confirming ESBLs.
We thank the following people who participated in the collection of isolates; Mrs Gbadeyan O. Felicia of Obafemi Awolowo University Teaching Hospital Complex, Ile-Ife, Osun state, Mr Adegoke of University College Hospital, Ibadan, Oyo state and Mr Akinbo of Federal Medical Centre, Ogun state. We also thank Dr Soge O. O. for his professional contributions. N. Mendonça was supported by a grant SFRH/BPD/45815/2008 from Fundação para a Ciência e a Tecnologia, Lisbon, Portugal. This work was supported financially by the ESCMID 2010 Research Grant from the European Society of Clinical Microbiology and Infectious Diseases and by the Center for Pharmaceutical Studies, University of Coimbra.
Sequencing analysis of the blaCTX-M detected in the four isolates identified the blaCTX-M-15 variant.
80
Bacterial isolates
% Distribution of isolates by type betalactamases produced
imipenem or tygecycline.
45 25
40
REFERENCES
35
Percentage (%)
20 30
Percentage (%)
ACKNOWLEDGMENTS
PCR detection of beta-lactamase genes in the ESBLproducers revealed blaTEM-like beta-lactamase in 24.2% and blaCTX-M in 3.3% of the isolates. The blaSHV was not detected in any of the isolates. Two of the blaCTX-M were associated with the ISEcp1 insertion sequence.
70
METHODS
The clinical isolates showing positive result for ESBLs were screened by PCR for the most common beta-lactamases: TEM, SHV, CTX-M and associated insertion sequences. The blaCTX-M amplicons were sequenced and identified using BLAST software.
Tigecycline, an antibiotic not in use in Nigeria, showed >90% antibacterial activity as well as imipenem against the ESBL-producers.
20
INTRODUCTION and OBJECTIVE
PCR and Sequencing
Of the 120 clinical isolates of Proteus spp. collected (45.8% male, 54.2% female), 91.7% were identified as P. mirabilis and 8.3% as P. vulgaris.
100
25 20 15
15
10
10 5 5 0
0 Wound
Urine
Ear
Eye
Pathological sources
ECS
Per. Efflu.
blaTEM
blaCTX-M
Beta-lactamase genes
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