Characterisation of Extended-Spectrum b-Lactamases among Multidrug Resistant Enterobacteriaceae from Sudan

The aim of the present study was to characterize extended-spectrum b-lactamase (ESBLs) genes in multidrug resistant enterobacterial pathogens as well as commensal isolates from the Sudan during the period 2003 to 2007. ESBL production was determined phenotypically by the combined disc method, and was characterized genotypically by the detection of blagenes by PCR and nucleotide sequencing. Transferability was examined by conjugation with nalidixic-acid resistant E. coli K12. The results showed that a total of 106 of the 113 (94%) isolates including E. coli, Klebsiella pneumoniae, proteus spp., Enterobacter cloacae, Providencia spp. and Morganella morganii, were positive for blagenes including the prototype blaTEM. Eleven isolates (28%) of the 113 were ESBL producers encoding blaSHV genes (SHV5, SHV5a, SHV12, SHV26, SHV28 and SHV38), 90 isolates (80%) were CTX-M positive. All, but only one (CTX-M9) were CTX-M15. Only 3(2.7%) of the isolates were Amp-C producers (CMY-4 and DHA-1). Plasmid transfer of the multiple resistance patterns was achieved among all the isolates. These findings demonstrated that ESBLs were highly produced by multi-resistant enterobacterial isolates from the Sudan; among both clinical pathogens as well as stool commensals. This is the first report of ESBLs genes characterization from the Sudan.


INTRODUCTION
Extended-spectrum b-lactamases (ESBLs) are b-lactamase enzymes capable of conferring bacterial resistance to the broad-spectrum oxyiminocephalosporins. They hydrolyse extendedspectrum cephalosporins, as well as aztreonam. They are inhibited by b-lactamase inhibitors such as clavulanic acid 1,2 . ESBLs are not active against cephamycins and most strains expressing ESBLs are susceptible to cefoxitin and cefotetan. However ESBL producing strains can become resistant to cephamycins due to acquisition of plasmidmediated Amp-C b-lactamases or to the loss of outer membrane porin proteins 3 . Carbepenems are less susceptible to hydrolysis by ESBLs than any other b-lactam antimicrobials, although resistance has been reported among mutants with porin loss 3 .
ESBLs represent a major health problem worldwide 4,5 . Over 60% of bacterial nosocomial isolates mostly E. coli and Klebsiella spp are claimed to be capable of beta-lactamase production 6,7 . ESBL-related infections are on the rise and has been observed throughout the globe on both hospital and community settings 8,9,10,11 . Communityacquired infections or colonization with ESBLproducing E. coli, were reported in several studies 12 as an important cause of community onset urinary tract infections 13 , as well as blood stream infections 14 . Multi-drug resistant E. coli 15,16 and ESBL producing Enterobacteriaceae have been reported from different hospitals in Sudan 17,18 . Recently, Musa has demonstrated predominance of blaCTX-M gene among ESBL producing E.coli and Klebsiella spp from Sudan 19 . We have undertaken this study in order to determine the prevalence and mechanism of antimicrobial resistance among Enterobacteriaceae from Sudan.

MATERIALS AND METHODS Bacterial strains
This study was carried out in two major tertiary care referral hospitals in the State of Khartoum (Omdurman and Khartoum teaching hospitals) in order to determine the prevalence and mechanisms of antimicrobial resistance among Enterobacteriaceae from Sudan. Clinical isolates were collected from patients and healthy individuals during the period 2003-2007. Two hundred clinical isolates, were obtained from each hospital Microbiology Laboratory and 121 normal fecal strains from inpatients and 500 fecal strains from healthy individuals in the community were included in the study. All 1021 bacterial strains were identified to the species level by standard microbiology methods 20 and confirmed by API-20E and were stored at -20°C in Cryopreservers until tested.

ESBL-Screening test
Bacterial strains resistant to ceftazidime and/or cefotaxime (113) were further screened for the production of Extended Spectrum Beta-Lactamases (ESBLs), according to BSAC guide lines 23 . Screening test was carried out using cefpodoxime (10µg) and cefoxitin (30µg) (Oxoid) antibiotic discs. Bacterial strains that were resistant to cefpodoxime were considered as ESBL producers while resistance to Cefoxitin indicates Amp-C production 23 .

ESBL-Confirmatory test
The Bacterial strains (113) were then subjected to the confirmatory test by the combined disc method. Three sets of antibiotic discs (Mast Laboratories Ltd, Bootle, Merseyside, UK) were used 23 .

Plasmid analysis and conjugation studies
Conjugal transfer of resistance determinants was performed by broth culture with E. coli K12 as a recipient. After 24 hours incubation, mating mixtures were plated onto agar supplemented with nalidixic acid (30µg/ ml) and ampicillin (10 µg /ml). Plasmid DNA was prepared from donors and transconjugants using a commercial kit (Plasmid Mini Kit, Qiagen GmbH, Hilden, Germany).

PCR amplification and sequencing
DNA for PCR was extracted by boiling of suspended bacteria on sterile water for 10 minutes. PCR was carried out for the identification of the bla genes coding for extended spectrum b-lactamases (TEM, SHV, and CTX-M) and for Amp-C Beta-Lactamases. PCR products were detected by electrophoresis on 1.5% (w/v) agarose gels visualized under UV light. Sequence determination of amplicons was performed using the respective PCR primers for both directions using ABI 3100 automated sequencer (Warrington, UK) and compared on BLAST.

Statistical Analysis
Results were analysed using SPSS software program.

Ethics approval and consent to participate
Not applicable, all study samples (isolates) were obtained from microbiology laboratories.
Screening of all the isolates (113) with Cefpodoxime showed that resistance among 111(98%) of the isolates.
Over all 106(94%) isolates were confirmed by the Combined disc method for ESBL production while 7(6%) isolates were tested negative ( Table  1).

ESBLs-producing strains
For the characterization of ESBL only multidrug resistant strains were resistant to cefotaxime or/and ceftazidime antimicrobials were included. The total number was One-hundred and Thirteen strains (113). Consisted of 103 (91%) clinical isolates and ten stool commensals (9%), eight of which were from hospitalized patients and two were from healthy individuals (table-2). bla genes were detected by PCR in 94% of the isolates (106/113), it includes different enterobacterial species comprising E.coli, Klebsiella pneumoniae, Proteus spp, Enterobacter spp, Salmonella spp and few others. Some isolates were found to possess more than one type of ESBL as well as the prototypic TEM-1 b-lactamase enzyme ( Table 3).    (Table 4). DNAsequencing results for blaTEM positive isolates revealed blaTEM-1 prototype.    24,25,26 . The most frequently encountered ESBL genes were CTX-M. This is consistent with the findings of Musa and Yousif from Sudan. However, our study was more detailed as we characterized CTX-M genes further and found that they are consistent mostly of CTX-M15. The CTX-M15 enzyme belongs to the CTX-M-1 cluster and is derived from CTX-M-3 by a single Asp-240-Gly substitution 27 and has the highest catalytic activity against ceftazidime among all the CTX-M clusters, this explains the high resistance level to Ceftazidime among this group of our isolates. CTX-M15 has been reported from different parts of the world, it has been described in many genera of enterobacteriacae across the world, and is the most reported variant in much of Europe 28 . We identified CTX-M15 in a number of Enterobacterial species, including E.coli, Klebsiella spp., Proteus spp., Enterobacter spp., Citrobacter freundii and Morganella morganii. All these strains were resistant to multiple antimicrobial agents. CTX-M15 variant was also detected in fecal commensal isolates in this study, two E.coli isolates from the community and six isolates including E.coli and Klebsiella spp from the fecal samples of hospitalized patients. The CTX-M-15 variant which occurs mainly in E.coli seems to be epidemic in Resistance transfer to the expandedspectrum cephalosporins was achieved among all the tested isolates, as well as the resistance to the other antimicrobial groups which was also co-transferred to the recipient organisms.

DISCUSSION
We have undertaken this study in order to find out the Prevalence of ESBL-producing Enterobacteriaceae among hospitalized patients and healthy subjects in the community and to characterize ESBL genes, as the first study of its kind in Sudan. The prevalence among Klebsiella spp., E.coli, Proteus spp. and Morganella morganii isolates from hospitalized patients was exceptionally high when compared to that from the community. Similar high ESBL prevalence among Enterobacteriaceae in Sudan was reported most countries. It is reported from many countries within the African continent with other ESBLenzymes. From Tanzania in a pediatric hospital 29 and an intensive care unit 30 , from Cameroon 31 . Another report from sub-Sahara Africa was from Central African Republic 32 , Malawi 26 . And also North Africa from Tunisia 33 .
Conjugation studies from this study have shown the possibility of horizontal gene transfer between various enterobacterial species carrying resistance genes to many antimicrobial groups including b-lactams. This could explain the acquisition of CTX-M15 and other ESBLs genes by different species of enterobacteriaceae. Other studies has reported similar findings, from Nigeria 34 , and Malawi 26 , showed that ESBL genes were associated with large conjugative plasmids that carry other resistance determinants and were responsible of resistance transfer between different bacterial species.
Only one isolate (E.coli) from this study was encoding blaCTX-M9, reported and sequenced in an E.coli from Spain 35 and also later reported in an E.coli causing community acquired UTI infections 36 .
Variable blaSHV genes encoding ESBLs have been detected in this study from clinical as well as commensals. blaSHV5 and blaSHV5a were detected in 9.7% of the ESBL producing isolates, both genes were isolated from Enterobacter spp and from K. pneumoniae. SHV5a was detected in an E.coli isolate from the community commensal collection as well. SHV5 has been reported by different studies from Thailand 37 , Poland 38 , Australia 39 and was isolated from a healthy student in Lebanon 40 . From the African continent SHV-5 has been reported from South-Africa in two different studies 41,42 . blaSHV12 was detected in two (2.7%) isolates (Klebsiella pneumoniae and an Enterobacter spp); both isolates were CTX-M15 producers. blaSHV12 is the most commonly identified genotype worldwide. within the African continent SHV-12 was reported from Tanzania 29,31 , Senegal 43 , Cameroon 32 and from the Central African Republic 33 .
blaSHV genes with specific hydrolytic activities to certain antimicrobials were also detected. SHV-38 was detected in Klebsiella and an Enterobacter isolates, its hydrolytic activity includes only ceftazidime and imipenem. It has been reported from France 45 . SHV-26 was also detected in this study in two K. pneumoniae isolates, they were resistant to co-amoxyclav. SHV-26 is a b-lactamase with reduced susceptibility to clavulanic acid that renders bacteria intermediately resistant to an inhibitor containing b-lactam.
Other blaSHV genes were also reported by this study including blaSHV-28 among two isolates K. pneumoniae and E.coli.
And SHV-11 which infrequently reported and is not considered as an extended spectrum b-lactamase 44 . The prototypes to TEM and SHV-1/SHV-1a b-lactamase enzymes were detected as well among the majority of the isolates.
This part of the study has illustrated a great variety of beta-lactamases genes, which reflects the magnitude of the problem of antimicrobial resistance in Sudan, and the wide misuse of the expanded-spectrum beta-lactams. Over all 69% of the isolates were carrying blaCTX-M gene as well as blaSHV gene. 31% were carrying only blaSHV genes and were all ESBL-producers.
Detection of CTX-M15 and the variety of SHV enzymes among the majority of isolates would render all third generation cephalosporins ineffective for the treatment of enterobacterial infections. Finding of Amp-C within these isolates was an alarming sign of the development of resistance to cephamycins which have not yet been introduced into clinical practice in Sudan. All the above would limit the treatment options for the physicians who would be faced with only one option. Imipenem has recently been introduced in The Sudan. However all the isolates showed complete susceptibility to it and it could be one of the options for the treatment of multi-resistant infections. However already a resistance gene (blaSHV-38) that confers resistance to ceftazidime as well as to imipenems has been detected among these isolates.
Resistant bacteria may also spread and become major infection-control problems. Not only within health-care institutions, but also in the community. Clinically important bacteria, such as extended-spectrum b-lactamase (ESBL)-producing E.coli, are increasingly observed in the community as has been reported in several studies 37,46 .
In this study, multiple drug resistance to three or more antimicrobials is highly prevalent among the clinical isolates found in variable enterobacterial species. A wide range of gramnegative bacterial species were found to be harboring resistance to the majority of commonly used b-lactam antibiotics as well as to other antimicrobial groups. Infection with multiresistant pathogens will complicate the treatment of nosocomial gram-negative bacterial infections within the Sudanese hospitals and will increase the mortality rates due to ineffective treatment of patients complicated by the limited options available for the doctors. This will result in further nosocomial dissemination of multi-resistant isolates due to selective pressure applied by the use of these drugs.

Compliance with Ethical Standards
This research is fully sponsored by Ahfad University for Women, Sudan.
E t h i c s o f h u m a n a n d a n i m a l Experimentation Not Applied.