Activity of beta-lactam antibiotics and production of beta-lactamases in bacteria isolated from wound infections in Brazzaville , Congo

To determine the mechanism of bacterial beta-lactam resistance, 165 bacteria isolated from wounds of hospitalized patients composing of: 42 Staphylococcus aureus, 37 Pseudomonas aeruginosa, 23 Escherichia coli, 22 Proteus, 12 Klebsiella, 10 coagulase-negative staphylococci (CNS), eight Enterobacter, six Citrobacter, five Providencia were tested for their sensitivity to beta-lactams and their production of beta-lactamases. The antibiotic susceptibility was considered by the method of the standard diffusion on agar Mueller Hinton. The rate of production of ß-lactamase in all bacteria was determined using the Strips of nitrocefin. The percentages of resistance to beta-lactams obtained were as follows: Staphylococcus aureus (77.90%), Pseudomonas aeruginosa (44.14%), E. coli (73.8%), Proteus (57.4%), Klebsiella (63.6%), CNS (57.15%), Enterobacter (56.3%), Citrobacter (83.3%), Providencia (67.5%). The rate of beta-lactamases were as follows: S. aureus (7.34%), P. aeruginosa (89.19%), E. coli (95.65%), Proteus (86.36%), Klebsiella (91.67%), CNS (90%), Enterobacter (87.5%), Citrobacter (66.67), Providencia (100%). The studied bacteria produce beta-lactamases which is the primary mechanism of resistance to beta-lactam antibiotics in the majority of the bacteria. Beta-lactamases rates vary from one genus to another. It is extended spectrum beta-lactamase-producing strain.


INTRODUCTION
Since the discovery of penicillin 80 years ago, Gramnegative bacteria have become proficient at evading the lethal activity of β-lactam antibiotics, principally through the production of β-lactamases (Rapp and Urban, 2012).Beta-lactam antibiotics are a broad class of antibiotics, consisting of all antibiotic agents that contain a β-lactam ring in their molecular structures.This includes penicillin derivatives (penams), cephalosporins (cephems), monobactams and carbapenems (Holten and Onusko, 2000).
They are a family of first-line antibiotics both in terms of availability and in terms of the cost.Since their discovery by Flemming in 1929 with penicillin, they are commonly used in the treatment of various infections (Livermore and Brown, 2001).Their use is of therapeutic importance, in the improvement of the infectious diseases treatment, the reduction of mortality and the eradication of the once formidable diseases.However, since their introduction in medicine, bacteria have developed mechanisms that have resulted in the emergence of the multi resistance.Several biochemical or genetic mechanisms are responsible for this resistance, including the production of enzymes (Andremont et al., 1997;Zogheib and Dupont, 2005).The infections caused by extended-spectrum beta-lactamase (ESBL) producing bacteria, constitute severe problems (Kuzucu et al., 2011).
The purpose of this study was to assess the rate of resistance of these bacteria to beta-lactams and determine the possible mechanism of inhibition of these antibiotics.

Strains identification
Gram negative bacteria which belong to the enteric bacteria group were identified and used in a leminor galery which comprise ureaindole, Kligler Hajna, citrate Simmons.The identification was confirmed by the tests of ONPG and sugar fermentation.
For pyocyanic bacilli, enhancing pigment production galery was assayed with the King A and B media.
In the genus staphylococcus, Gram positive bacteria were isolated and identified by using cultural and biochemical characters using Chapman media (production or not of yellow pigment).Identification was confirmed by the tests of catalase and coagulase.

Strains susceptibility
Betalactam antibiotics susceptibility was determined by disk diffusion on Mueller Hinton (Sanofi pasteur) medium (Carret et al., 2001;Esmaeili et al., 2014).Discks of beta-lactam antibiotics have been used in natural resistance to each type or kind of bacteria.The method, Etest was also used as described by Saito et al. (2013).
The beta-lactamases were detected using a chromogen substrate, laboratories Oxoid nitrocefin.A colony of a growing strain was put in contact with the substrate on the strip of nitrocefin.After 5 to 10 min of contact, two (2) drops of reagent were added to the nitrocefinase.Hydrolysis by beta-lactamase substrate results in the development of a red color with a blue ring on the disk, which reveals the complex enzyme-substrate (Spicier, 2001).The betalactamase distribution was analyzed by excel.

RESULTS
The activities of the beta-lactam antibiotics tested on the staphylococci are given in Table 1 and those of the Enterobacteriaceae are given in Table 2.For the Pseudomonas, the rate of resistance to beta-lactams obtained is 44.14% with total inhibition of carbenicillin.Ceftazidime and the imipeneme were respectively inhibited by 21.62 and 10.82% of strains.The numbers of positive strains to the test of nitrocefin for each group of bacteria as well as the rate of beta-lactamases are respectively given in Table 3 and Figure 1.

DISCUSSION
Resistance of Staphylococci to tested beta-lactam antibiotics is not very high.These results are consistent with those given in the literature by several authors such as Aryam et al. (2005) and Tronel et al. (2002).These results can be explained by their use as first-line antibiotic.The meticillino-observed resistance is due to the acquisition and integration of the mecA gene, which induces the synthesis of the PLP2a which is able to assemble Peptidoglycan with low affinity for oxacillin (methicillin) and other beta-lactam antibiotics (Fatholahzadeh et al., 2008;Benoit et al., 2013).
In enteric bacteria, the imipeneme and ceftazidime have been the most active betalactams.These results are consistent with that of Sotto et al. (2001) and Lagacé-Wiens et al. (2014).The sensitivity rates of these strains to ceftazidime, were compared with those obtained by Petra et al. (2007).
The association of amoxicillin with clavulanic acid has been completely inactive in some Enterobacteriaceae, and the other has presented a high resistance.This result can be explained by the modification of penicillin (PLP) binding protein which confers resistance to most of the betalactam antibiotics (Sotto et al., 2001).The cephalosporin's resistance may be due to the production of cephalosporinases as suggested by Bertrand and Talon (2001).In most cases, the mechanism of resistance to beta-lactams in Enterobacteriaceae is the production of betalactamases (Bedenic, 2004).
With regards to Pseudomonas, among the tested beta-lactam antibiotics, total resistance to carbenicillin was observed.This result is different from those obtained by Yu et al., (2006) which demonstrated that carbapenems remain the drugs of choice for serious infections caused by  ESBL-producing organisms.
The ceftazidime was inactivated by 21.62% of strains and two strains of intermediate sensitivity were observed.This level of sensitivity presents a slight difference with the data of Sevillano et al. (2006).The appearance of resistance to ceftazidime in Pseudomonas may be a marker of the multi resistance for hyper-producing betalactamases strains which represents a factor of risk for the emergence of these strains (Cavallo et al., 2003).
The imipeneme has been the most active antibiotic with only 10.82% of resistant strains.These results are consistent with those obtained by Sinave (2003) and Soussy (2001).The development of resistance to the imipeneme is a risk factor for emergence of strains of Pseudomonas multi resistant strain (Rossilini and Mantengolie, 2005).However, high rates of resistance to the imipeneme and ceftazidime on hospital strains were obtained by Sekowska et al. (2005).
Most of the tested bacteria had shown high levels of beta-lactamases.These results have confirmed the work of Susić (2004), Akujobi (2005) and Bedenic (2004).The low rate obtained in Staphylococcus aureus, is explained by the resistance to oxacillin linked to the acquisition of the mec A gene.With regards to the strains of Enterobacter, beta-lactamases rates are comparable to those reported by Petra et al. (2007); Koren and Vaculíková (2006).These beta-lactamases can spread more often by the interspecific transfer of resistance between the cocci Gram positive and bacilli Gram negative by conjugation.
Tested Bacteria Percentages of beta-lactamases

Table 1 .
Activity of beta-lactam antibiotics on staphylococci.

Table 2 .
Activity of beta-lactams in Enterobacteriaceae.

Table 3 .
Distribution of positive beta-lactamase strains.