The threat to human health posed by antibiotic resistance is of growing concern. Many commensal and pathogenic organisms have developed resistance to well established and newer antibiotics. Although a number of factors can be identified which contribute to the problem, clearly the antibiotic as a selective agent and the resistance gene as the vehicle of resistance are the two most import factors. Antibiotic use has encouraged growth of the resistant strains, leading to an imbalance in prior relationships between susceptible and resistant bacteria. In Pakistan, like the other developing countries, there is a general increase in the antibiotic resistance as the availability and use of antibiotics are poorly controlled, which results in a high rate of resistance, particularly to the older antibiotics.
The study was carried out for the period of two years from January 1998 to December 1999, in order to evaluate the prevalence and development of resistance in clinically significant gram-negative bacilli against commonly used antibiotics. Bacterial growth was identified up to genus and species level. A total of 8,638 samples were received by the pathology laboratory of Pakistan Institute of Medical Sciences, Islamabad, Pakistan, which comprised of 5,373 pus, 2,188 blood and 1,077 miscellaneous samples.. Miscellaneous group represented samples which comprised of few specimens and included sputum, urine, stool, different body fluids, high vaginal swabs, ear/throat/nasal swabs, catheters, tracheal secretions and wounds. Miscellaneous samples were evaluated during 1999 only, while pus and blood were studied in both the years.
E. coli was found to be the most prevalent gram-negative bacilli from all groups, in both the years. Gender-wise prevalence, revealed a trend of high infection rate in males. In pus samples 70.6% male patients were infected with E. coli while in case of P. aeruginosa 76.4% and 23.6% patients infected were males and females respectively. Similar trend of higher infection rate in males was also observed in all other gram-negative bacilli. An identical pattern of high number of infected males among bacilli was also noticed in blood samples and miscellaneous samples, except where 65.8% female patients were infected with E. coli in miscellaneous samples. The data of month-wise prevalence for pus samples did not showed any definite trend, occurrence of various gram-negative bacilli was scattered all over the year in both 1998 and 1999. The isolates were subjected to culture sensitivity test using Bauer and Kirby disc diffusion method. Overall 38 antimicrobials were used during the study period from January 1998 to December 1999.
In case of E. coli high resistance was observed for ampicillin (94.2%), K pneumoniae also showed highest resistance against ampicillin (95.0%), whereas, P. aeruginosa exhibited maximum resistance to carbenicillin (83.4%). Among most commonly used antibiotics, piperacillin proved to be most effective against P. aeruginosa with resistance rate of only 3.4%. In this study, among most frequently used antibiotics discs, Pseudomonas spp., exhibited highest resistance to carbenicillin (83.2%). Piperacillin was found to be most effective, with the resistance rate of only 4.6%. The study also demonstrated that Enterobacter spp., was found to be highly resistant to ampicillin with a rate of (82.4%). While analyzing cumulative usage of different antibiotic groups for susceptibility testing, the highest cumulative usage was noticed in cephalosporins, followed by penicillins, aminoglycosides and quinolones, respectively.
Minimum inhibitory concentration (MIC) values of E. coli (n=112), K pneumoniae (n=36) and P. aeruginosa (n=44) were determined using agar dilution method for all the 192 isolates of these three species of gram-negative bacilli randomly selected from clinical samples. MIC values were determined against four antibiotics including ampicillin, cephradine, gentamicin and ciprofloxacin, representing the four groups of antibiotics penicillin, cephalosporins, aminoglycosides and quinolones respectively. For each antibiotic along with the active pharmaceutical ingredient (API), four brands (injectables) which were available in the market were also used for MIC determination, except for ciprofloxacin, where only three brands were available in injectable form.
MIC50 and MIC90 values for E. coli, K pneumoniae and P. aeruginosa against ampicillin, cephradine, gentamicin and ciprofloxacin, and their brands, were also determined and subjected to Student 't' -test, Correlation and Single Factor Analysis of Variance (ANOV A). The isolates were divided into two categories, â€œPrimaryâ€, which contained all isolates, including isolates whose MIC50 and MIC90 values determined were <16 Âµg/mL, and >2048 Âµg/mL. â€œSecondaryâ€, which comprised of only those isolates whose MIC values fell between 16 to 2048 Âµg/mL, except for the ciprofloxacin, where the range was 1-256 Âµg/mL.
When MIC50 and MIC90 values of E. coli, K pneumoniae and P. aeruginosa against ampicillin (API) and its four brands were compared by ANOV A in case of both primary and secondary categories, the p-value was found to be significant (p<0.05) in all the cases, indicating that significant difference existed among MIC50 and MIC90 values of ampicillin (API) and its four market brands against E. coli, K pneumoniae and P. aeruginosa.
Significant difference (p<0.05) was observed between cephradine (API) and its brands with reference to MIC50 values among primary and secondary categories of E. coli, and only primary category of P. aeruginosa through application of ANOV A. With reference to MIC90, significant difference (p<0.05) was found in primary groups of E. coli and P. aeruginosa, whereas K pneumoniae in this category showed non significant difference (p>0.05). Non significant difference (p>0.05), with reference to MIC50 was seen in primary and secondary categories of K pneumoniae and only in secondary category of P. aeruginosa. Similarly, a non significant difference (p>0.05) with reference to MIC90 value was also observed among primaryand secondary categories of K pneumoniae and in secondary category of P. aeruginosa only.
When MIC50 and MIC90 values of ciprofloxacin and its three market brands and gentamicin along with its four brands were analyzed statistically using single factor analysis of variance, no significant difference (p>0.05) was observed between ciprofloxacin (API), gentamicin (API) and their market brands against both primary and secondary categories of E. coli, K. pneumoniae and P. aeruginosa isolates.
Minimum bactericidal concentrations (MBC) using broth macrodilution method were determined for those isolates of E. coli, K. pneumoniae and P. aeruginosa whose MICs were fell between 16 to 2048 Âµg/mL while testing with agar dilution method against ampicillin, cephradine, and gentamicin (APIs) and their market brands, and 1-256 Âµg/mL for ciprofloxacin (API) and its market brands.
The overall MBCs of ampicillin, cephradine, gentamicin and ciprofloxacin against E. coli, K. pneumoniae and P. aeruginosa ranged from 1 to 2 two-fold dilutions more than their MICs
Isolates of E. coli (n=112) and K. pneumoniae (n=36) from clinical samples were screened using double disc diffusion method to determine the prevalence of ESBL-producer. Among E. coli 14 (12.5%) while in case of K. pneumoniae 13 (36.1 %) were found to be ESBL-producers. ESBL-producing strains were resistant to most of the Î²-Iactam and non Î²-Iactams, but a majority of them were still susceptible to imipenem.
The study revealed that antibiotic resistance has become a significant problem and will continue as bacteria continue to evolve under the selective pressure of antibiotics. It can be controlled with continued surveillance, infection control procedures and improved antibiotic usage, which are critical in preventing the reemergence of infectious diseases as a major cause of morbidity and mortality.