Antimicrobial resistance status and prevalence rates of extended spectrum beta-lactamase producers isolated from a mixed human population

Owing to the increasing epidemiological and therapeutic challenges associated with infections due to ESBL producers, ESBL prevalence rate among some bacteria isolates from healthy and non-healthy human population in a metropolitan Nigerian setting was evaluated. A total of one hundred and forty-fi ve () bacteria strains were isolated from a total of four hundred and sixty () samples collected from urine, wound, throat and anal swabs of  healthy volunteers in the community and from  patients in  secondary and  tertiary hospitals (altogether, ) in Enugu metropolis. Th e presumptive confi rmatory test used for ESBL detection was the Double Disc Synergy Test (DDST) method. Conjugation and plasmid curing studies were also done for resistance factor determination. Of the  isolates,  were ESBL producers with  of these ESBL producers being of community origin and  from hospitals. Th is translates to . and  incidences (comparably higher than established prevalence of . and . respectively) for community and hospital infections respectively. Th e ESBL isolates showed high resistance to tetracycline, gentamicin, pefl oxacin, ceftriaxone, cefuroxime, ciprofl oxacin and Augmentin® (Amoxicilin and clavulanic acid combination). Conjugation studies for Resistance plasmid transfer showed non-transference of resistance determinants between the ESBL transconjugants and recipient strains. Correspondingly, the plasmid curing studies revealed that the acridine orange could not eff ect a cure on the isolates as they still retained high resistance to the antibiotics after the treatment. Th is study confi rms the growing incidences/pool of ESBL strains in Nigeria and call for widespread and continuous monitoring towards an eff ective management of the potential therapeutic hurdle posed by this trend. ©  Association of Basic Medical Sciences of FBIH. All rights reserved


INTRODUCTION
Th e introduction of third-and fourth-generation cephalosporins as therapeutic agents has been followed by the dissemination of diff erent extended-spectrum b-lactamases (ESBLs) that hydrolyse those b-lactam drugs and also monobactam antibiotics.ESBL are bacterial enzymes that hydrolyse and confer resistance to modern cephalosporin antibiotics.Th ey constitute the major mechanism of resistance to second, third and fourth generation cephalosporins (for example: cefuroxime, cefotaxime, ceftriaxone and ceftazidime) [-].Additionally unfortunate is the discovery that ESBL-producing organisms often also possess resistance determinants to other important antibiotic groups, such as aminoglycosides and fl uoroquinolones, leaving an extremely limited range of eff ective agents [].ESBLs have been found in a great number of diff erent bacterial species, but more frequently in Escherichia coli and Klebsiella pneumoniae [, ].Th ere have also been reports of the growing concern of the Enterobacteriaceae and Pseudomonas spp producing extended-spectrum b-lactamases (ESBLs) among nosocomial and also community-acquired infections [-].Clinicians, microbiologists, infection control practitioners, and hospital epidemiologists are concerned about ESBL-producing bacteria because of the increasing incidence of such infections, the limitations of eff ective antimicrobial drug therapy, and adverse patient outcomes [, -].In Nigeria, there have been reports of the reoccurring cases of antimicrobial resistance by most pathogenic organisms against many antibiotics [].Moreover, fractional isolated studies establishing the presence of ESBL producing bacteria clinical isolates from specifi c localities within the Western and Eastern part of the country have also been reported [-].Th ese initial preliminary reports have further heightened the necessity of extending similar studies to cover more unsampled localities, to further harmonize the epidemiological data base, and the need for a continuous epidemiological monitoring of the prevalence rate of these ESBL bacteria isolates.Data generated from such foregoing exercises would not only defi ne the existent bacteria resistance indices but also clearly serve as a useful baseline in determining the rational and eff ective chemotherapeutic options for the management of infectious disease due to the ESBL bacteria organisms.It is therefore based on these established premises that this present study was carried out to determine the prevalence rate of these pathogenic ESBL-producing enterobacteriaceae organisms in the sampled community.

Microorganisms
Samples were collected over a fi ve months period between October  and February .Informed consent and ethical approval was obtained.A total of four hundred and sixty () samples (urine (), vaginal (), anal (), wound (), and throat swabs ()) were collected.Two hundred and twenty () samples from , , and  healthy human volunteers in the community aged -, -, and - years respectively, while two hundred and forty samples () from  and  patients aged -, and - respectively from four () hospitals comprising University of Nigeria Teaching Hospital (UNTH); Enugu, National Orthopaedic Hospital, Enugu (NOHE), Ntasiobi Ndinona Afufu (NONA), and Reego Laboratories, Enugu.Sixty eight () and one hundred and seventy-two () samples from hospital patients were collected below and over  hours post-admission respectively.Characterization of isolates was according to recommended standard technique by the National Committee for Clinical Laboratory Standard (NCCLS).

Antibiotic Sensitivity test
Antibiotic sensitivity of the isolates was determined using previously established procedure [].Briefl y, the isolates were cultured in nutrient broth at   C for  h.Two () loopfuls of the suspension of each isolate were inoculated into ml of sterile molten agar in  cm diameter Petri dishes and mixed.Th e plates were allowed to set and the antibiotic Sensitivity discs were aseptically placed on their surfaces.Th e plates were incubated at   C for  h and the resultant inhibition zone diameters (IZDs) were measured and recorded.

Test for ESBL Production
Th e isolates were tested for ESBL production using the double disc synergy test (DDST) method. .A combination disc (Amoxicillin  μg and Clavulanic acid  μg) was placed at the centre of the Petri dish and antibiotics (Ciprofl oxacin  μg and Cefuroxime  μg) were placed  mm apart on both sides of the plates.It was incubated at  o C for  hours after which the various inhibition zone diameters were measured.

Resistance Transfer
Resistance transfer to ascertain if the resistance determinants were plasmid mediated was determined by conjugation test.ESBL positive isolates of Klebsiella pneumoniae, Klebsiella oxytoca, Escherichia coli and Pseudomonas aeruginosa were mated with an Escherichia coli recipient strain that is rifampicin resistant and susceptible to Antibiotic Resistant Markers (ARM) that will include gentamicin, ampicillin, tetracycline, chloramphenicol and trimethoprim/sulphamethoxazole.Recipient and donor strains were grown in Mueller Hinton broth at   C for - hr.Th e donor and recipient strains were mixed in a ratio of (:) and incubated for  hours at   C. Samples of  ml and  ml of donor to recipient mixture were spread-plated on the surface of MacConkey agar plates supplemented with  mg/ml of rifampicin and  mg/ml of ampicillin (oxoid U.K) and incubated at   C for  - hr.Samples from the donor and recipient were used as control.

Plasmid Curing
Further confirmatory plasmid curing studies were carried out to further confi rm the location of the resistance determinants.Briefl y, the organisms were grown at   C for  - hr in Mueller Hinton broth.The organisms were diluted to x  cfu/ml in Mueller Hinton broth and . mg/ ml of acridine orange was added to the  hr broth and incubated at   C for  hr.Th e cells were tested for plasmid curing by subjecting them to further antibiogram studies.

RESULTS
Table  shows the antibiogram of the hospital and community isolates used in the study.From the table, high resistance by the isolates to most of the antibiotics is refl ected.Over  of the community isolates were resistant to the antibiotics-Tetracycline, Augmentin ® (Amoxicillin and Clavulanic acid combination), ceftriaxone, ciprofloxacin, pefl oxacin, gentamicin, and cefuroxime while only about  of the hospital isolates were resistant to the same antibiotics.All the isolates however showed high susceptibility to imipenem.Also, Table   Conjugation studies showed no visible growth inhibition on any of the plates suggesting that none of the transconjugated organism could transfer its plasmid DNA to the recipient strain.Th e subsequent plasmid curing studies (Table ) reveal that the isolates still retained high resistance to the antibiotics after the treat-   ment with acridine orange, the implication being that the resistance determinant might not be extrachromosomal.

DISCUSSION
Antibiotic resistant bacteria have been a source of ever-increasing therapeutic problem [].Continued mismanaged selective pressure has contributed towards the emergence of multiple-drug-resistant bacteria and that has been regarded as an inevitable genetic response to misappropriated exposures of microbial populations to antimicrobial therapy [-].Th ese misappropriated exposures to a wide and varied antimicrobial cocktails no doubt has contributed to the troublesome bacteria resistance pattern recorded worldwide especially in the developing countries.In tropical areas, early-onset infections may be caused by multi-drug resistant (MDR) bacteria strains.Th ese organisms are usually resistant genera of the family Enterobacteriaceae, Pseudomonas spp., and Staphylococci [].In the present area under study also, we have equally identifi ed the MDR bacteria strains to belong principally to the Enterobacteriaceae family and Pseudomonas genera with a greater percentage skewing towards the Enterobacteriaceae category.Another closer observation of the results obtained seem to suggest a pressurized infl uence of mismanaged chemotherapy of nosocomial infections given the greater proportional position occupied by the hospital isolates where   of bacteria strains sampled were MDRhospital-isolates and only   being MDR-communityisolates (Table ).However, further observation of the data presented shows that the community isolates displayed more extensive resistances coverage to a wider cocktail of antibiotics utilized in the study.Th is fi nding does not appear to be fully understood since the community origin of the isolates does not normally seem to point to any previously known heavy antibiotic exposure.Th is consideration nevertheless may suff er credibility given the relatively poor and porous antibiotics distribution and use control existent in Nigeria that keep ensuring that individuals within the community have easy, abusive and uncontrolled access to antibiotics [].And in certain cases, these misguided individuals employ the use of high-action-profi le antibiotics in abusive proportions thus compromising the future positioning of this special category of antibiotics as treatment options of last resort in highly resistant bacteria infections.Th erefore, adequate drug and antibiotics control if pursued as an indispensable project by all stakeholders involved in healthcare delivery is expected to immensely contribute towards controlling this ugly trend.Moreover, given that the isolates were sensitive to imipenem and not to the other majority of the employed antibiotics is quite suggestive that they are Extended Spectrum Beta-Lactamase (ESBL) producing organisms.Th is assertion was further confi rmed by the double disc synergy test (DDST), thus leading to selection of  ESBL producing bacteria isolates among the sampled bacteria population were identifi ed (Table ).DDST for the detection and confi rmation of ESBL production involves the recording of increased susceptibility to at least one of the extended spectrum cephalosporins in the presence of a combination disc (amoxicillin/clavulanic acid combination) when placed mm apart centre to centre [].Th is means of detection of ESBL has been widely employed by many workers [-].Meanwhile, from our fi ndings, given that   of the confi rmed ESBL producers were identifi ed as Klebsiella spp (Table ) places this group in a luminous position suggesting its possibly major contributory role as a principal repository and transmitter of the putative extrachromosomal ESBL conferring determinants in the sampled area.Earlier reports of ESBL producing strains have showcased Klebsiella spp. as possessing a traditional role in the overall defi nition and expression of ESBL [].We have therefore the emerging problem of antibacterial chemotherapy that would involve an increasing plethora of ESBL-producing bacteria population strains in the tropics, and this poses a huge medical and economic challenge.It is equally important to recall that previous investigators working in Nigeria have reported related findings from neighbouring localities [, ].And recently, the ESBL hospital and community preva-    [, ] falling slightly short of the values obtained in our study.Th is increasing emergence and development of ESBL producing bacteria strains which remains a decimating therapeutic impediment clearly point to a present and troublesome problem that could constitutes a great deal of menace to futuristic infectious diseases control exercises.Hence, a great deal of attention is required to conduct studies to both identify and fully understand this problem -its cause and scope -so as to create an enabling and useful baseline for eff ective handling of the ESBL threat.Conjugation studies for Resistance plasmid transfer showed non-transference of resistance determinants between the ESBL strains and the transconjugants.Th is therefore suggests a possible chromosomal location of all recorded ESBL traits since chromosomally located genetic determinants cannot be transferred through non-replicating bacteria conjugation.In a corresponding similar outcome, the plasmid curing studies (Table ) also revealed that the acridine orange could not affect a cure on the isolates as they still retained relatively high resistances to the antibiotics after the treatment.Th is again would further corroborate the chromosomal location of the ESBL traits.However, an exception recorded was the singular isolated case of the Pseudomonas aeruginosa that swung from resistance status to sensitivity upon exposure to ceftriaxone (CRO) before and after the gene curing exercise.Th is nonetheless, the fi ndings from the twin tests of plasmid gene curing exercise and conjugation studies strongly point to the chromosomal loci of the resistance gene markers.When this is considered in relation to other previous reports of ESBL occurrence coming from Nigeria there seem to be a palpable variability among the ESBL-producing strains with respect to the chromosomal and extra-chromosomal location of the resistance determinants.Enabulele et al. [] reported that the observed resistance by gram negative wound isolates from the University of Benin were plasmid-mediated.However, in a later study Yah et al. [] working with ESBL-producing bacteria strains from Ahmadu Bello University Teaching Hospital (ABUTH) Zaria, reported their fi ndings that the bacteria population showed a distribution into sensitive and resistant post-plasmid-curing groups representing both chromosomal and extrachromosomal located resistance markers.Later, Iroha et al. [, ] accorded chromosomal location to the resistance determinants of the ESBL-producing bacteria strains they encountered.When these are considered in relation to our present fi ndings they seem to underscore the established variability in the cellular location of the resistance markers.Th is scenario, apart from determining the ease of passage and acquisition of the resistance traits, also, underscores the need for an extensive and constant demographic coverage of the country for an antimicrobial surveillance studies especially the specialized ESBL-producing bacteria strains.Th ese data would be useful for present and future intervention exercises.

CONCLUSION
The outcome of this study has demonstrated the palpable presence of ESBL producing bacteria strains from community and hospital subjects within Enugu metropolis.Th is kind of study remains relevant towards providing adequate baseline for the future projection and eff ective management of infectious diseases caused by the ESBL producing bacteria strains.

DECLARATION OF INTEREST
Th e authors declare that they have no competing interests.
DAMIAN C. ODIMEGWU ET AL.: ANTIMICROBIAL RESISTANCE STATUS AND PREVALENCE RATES OF EXTENDED SPECTRUM BETALACTAMASE PRODUCERS ISOLATED FROM A MIXED HUMAN POPULATION records that the identifi ed MDR-bacteria strains to be distributed largely between the Enterobacteriaceae and gin.Of the  bacteria strains identifi ed as ESBL producers,  ( ) were Klebsiella spp,  ( ) were E. coli while the remaining  ( ) were Pseudomonas spp.

TABLE 1 .
Antibiogram of hospital and community isolates from ENUGU Inhibition Zone Diameter of antibiotics (mm)

TABLE 2 .
Percentage resistance and sensitivity pattern of the isolates