The rapid and irrepressible increase in antimicrobial resistance of pathogenic bacteria that has been observed over the last decades is widely accepted to be one of the major problems of human medicine today. Several aspects of this situation are especially worrying. Many resistance mechanisms that emerge and spread in bacteria populations are those of wide activity spectra, which compromise all or a majority of drugs belonging to a given therapeutic group. Finally, multiple mechanisms affecting the same or different groups of antimicrobials coexist and are even co-selected in more and more strains of pathogenic bacteria. (Antimicrobial agents and Chemotherapy , March2012:vol. 56(3).

Enterobacteriaceae are short gram-negative rods,either motile or non motile,grow well on MacConkey agar, grows aerobically or anaerobically ,ferment rather than oxidize glucose often with gas production, are catalase positive, oxidase negative and reduce nitrate to nitrite. (Geo.F.Brooks,et.al (2010), Jawetz, Melnick & Adelberg’s Medical  Microbiology, 25th Ed. Pp 340).
One of the most resistant mechanism is that found in Enterobacteriaceae, which reduces the efficacy even of modem expanded – spectrum cephalosporin’s (except cephamycins and carbapenems) and monobatams is based on plasmid mediated production of extended spectrum beta lactamases (ESBL).
Extended spectrum beta lactamase producing strains have been detected to increase in the general community were studies have been published about its prevalence in healthy humans. (Antimicrobial Agents and Chermotherapy) volume 56.3 ).
Extended spectrum beta lactamases arises as a result of mutations in the TEM -1,TEM -2 or SHV-1 genes, commonly found in the Enterobacteriaceae family.These enzymes are found predominantly in klebsiella species and Escherichia coli.
They are capable of hydrolyzing penicillins,broad spectrum cephalosorins and monobactams but they do not affect the cephamycins or carbapenems and their activity is inhibited by clavulanic acid.
Klebisieua pneumonia and Escherichia coli are one group of beta lactamases (penicillin destroying enzymes) that produce the enzyme called extended spectrum beta lactamases(ESBLs),because they confer upon bacteria the additional ability to hydrolyze the beta lactam rings of cefotaxime,ceftazime or aztreonam.They are normal flora of the large intestine. The most common extended spectrum beta lactamases in three decades ago were the class A TEM and SHV plasmid mediated types. (Geo. F. Brooks, et al; (2010). Jawetz, Melnick and Adelerg’s Medical Microbiology (25thEd), Pp.340. McGraw Hill).
But the emergence of K. Pnevmonia carbapenemases (KPC) which confer resistance to third and fourth generation cephalosporins and carbapenems, led to plasmid mediated resistance mechanism. CTX-M enzymes have become more prevalent resistant throughout the world. (Geo. F Brooks, et al (2010). Jawetz, Melnick and Adleberg’s Medical Microbiology, 25th Ed, Pp. 340, McGraw Hill).

This is to screen for the occurrence of Extended spectrum beta lactamase producing Enterobacterianceae in healthy carriers in Abakaliki fast- food joints.

Routine samples will be collected from healthy staff members of fast-food joints in Abakaliki Urban areas. 300 samples (feaces) will be collected in sterile tubes. There will be no transport medium because the samples will be processed within 24 hours of collection. Each person will be tested once and the sample will be collected form staff without diarrhea. Their ages should be between 20 and 60 years, mainly females.

            Enterobacteriaceae are short gram-negative rods, either motile with peritrichous flagella or non-motile, grow well on McConkey agar, facultative aerobes or anaerobes), ferment rather than oxidize glucose, often with gas production, are catalase positive, oxidize negative and reduce nitrate. (Geo.F.Brooks,et al;(2010), Jawetz, Melnick & Adelberg’s Medical Microbiology (25th Ed). Pp. 213-214, McGraw Hill).
E.coli and K.pneumonia as my major organisms of study can thus be differentiated;
            E. coli forms circular convex, smooth colonies with distinct edges, motile, indole positive, citrate negative and voges-proskauer negative, acid fermenters and  methyl-red test (90%) where as k. pneumonia forms large and mucoid colonies which tent to coalesce with prolonged incubation, non-motile, indole negative, citrate and voges-proskaver positive, butanediol fermenters and methyl-red test (10%). (Geo.F.Brooks,et al; (2010),Jawetz,Melnick & Adelberg’s Medical Microbiology,25th Ed. Pp. 214-215, McGraw Hill).

Eosin-methylene blue (EMB), MacConkey or deoxycholate medium can be use as they contain special dyes and carbohydrates which distinguishes lactose-fermenting (coloured) from non-lactose fermenting (non-pigmented) colonies and allows rapid presumptive identification of enteric bacteria. Triple Sugar Iron (TSI) agar, will also be used as they help to differentiate salmonella and Shigella spps from the samples. Serotype will be performed according to the standard method. (Geo. F.Brooks,et.al; (2010),Jawetz,Melnick & Adelberg’s Medical Microbiology,25th Ed. Pp 214,McGraw Hill).

Disk diffusion method according to Clinical and Laboratory Standard Institute (CLSI) protocols will be performed and evaluated according to their criteria with 15 antimicrobial agents such as Ampillicin, cephalothin, cefuroxine, cefpodoxime, cofotaxime, ceftazidime, cefepime, trimethroprim-sulfamethoxazole, tetracycline, ciprofloxacin polymyxin, amoxicillin, clavulanic acid, cefoxitin, kanamycin and gentamicin.
ESBL producers will be confirmed on muller-Hintor agar plates using E test –ESBL-strips containing cofokaxime, cefepime or ceftazidime each alone and in combination with clavulanic acid.

Oxidase test will performed and non-fermented samples will be discarded. Indole test, methyline red, voges proskauer test, citrate test, motility test, manitol fermentation and hydrogen sulfide test will be carried out to enable distinguish E.coli from K.pneumonia.

The bacterial strains confirmed as producing ESBLS will be further analyze by PCR and by sequencing the whole open reading flames (ORF) of bla genes. DNA will be extracted by a standard heat lysis protocol.

Five specific published primer sets and PCR protocols (13,30,33,38) will be used to search for B-lactmase encoding genes belonging to blaTEM, and three blaCTX-M sub-families to ensure coverage of all entire bla ORFs. The primer sets will be supplemented with the following primers from up and down streams blaCTX-M flanking regions, such as;
The results will be purified using the PCR purification kit (according to the manufacturer’s recommendations).

Antimicrobial Agents and Chemotherapy, Volume 56(3).
Emerging infections disease, April 2001;Volume,7(2).
Emerging infections disease, Feb. 2008; Volume ,7

Geo F. Brooks, Karen C. Caroll, Janet S. Butel, Stephen A. Morse and Timothy A. Mietzner, (2010); Jawetz, Melnick & Adelberg’s Medical Microbiology 25th Ed, McGraw Hill).

Pathogenic Profile Dictionary, 2010. Undergraduate of Biological Sciences,

Science Gov.Com;Science indeed, www:science.gv/biochemical characterization bacteria.

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