Every year the clinicians write more than 160 million prescriptions in the United States alone, with subjects consuming 235 million doses of antibiotics annually of which only 50% – 80% is actually necessary.
Drug – resistant infections are more likely to cause longer hospitalizations to the affected, more severe side effects and incur more expenses.
A feverishly important question, doing its rounds in an era of growing microbial resistance is “how microbes manage to defeat antibiotics?” The answers are, as diverse as the problem itself. “Molecular biology is telling us . . . . what the resistant mechanisms are , although we don’t know all the details , “ says Julian Davies from University of British Columbia .
The common prevalent explanation is that bacteria in order to gain resistance to antibiotics, bacteria rely on mutations. Drug manufacturers in their endeavor to modify an existing antibiotic, impenetrable to resistant strains cause the bacteria to mutate and regain mastery over the new antibiotic, which already had a gene to defeat the older version. The following are the three commonly believed ways in which bacteria acquire genes for resistance:
1 . Spontaneous mutation: the bacterial DNA changes spontaneously, as in starburst. e.g :- Drug resistant tuberculosis .
2 . Transformation: A form of microbial sex wherein one bacterium takes DNA from another. e.g :- Gonorrhea (defined) resistant to Penicillin .
3 . Plasmid mediated resistance: A small circle of DNA called plasmid flits between different bacterial types carrying multiple resistances. e.g :- The case of Shigella diarrhea claiming lives of 12 , 500 Guatemalans in 1968 . The microbe harbored a plasmid resistant to four antibiotics.
The resistance of bacteria to antibiotics is not only attributed to “mutation”. Sharing of resistant genes also has a role to play. Plasmids — existing outside the main chromosomes as if they were mini chromosomes are also shared. In bacterial phylogeny (defined) this type of sharing is able to leap broad divisions.
There are genes called gene cassettes that can be integrated in the chromosomes (defined). Integrons which are genes code for integraces which can integrate the cassettes into chromosomes or other genetic material, where they start working making the integrons function like a carrier. Bacteria are able to pick up several cassettes and hence obtain resistance to several antibiotics. “Bacteria also integrate resistance to disinfectants or pollutants in these clusters, says Abigail Salyers of the university of Illinois.
The Microbial Mind :-
“The issue of resistance is converging from the human infectious disease and agricultural angles,” says plant pathologist Jo Handelsman, “whether the microbe is trying to protect itself against antibiotics, fungicides, insecticides, herbicides, even antiviral agents.” Handelsman, from the University of Wisconsin – Madison, points to more similarities. “At the molecular level, there are only a few mechanisms of resistance: change the target molecule, inactivate or decompose the drug or pesticide, sequester (defined) the drug or pesticide, or keep it out of the cell” to begin with. “We find resistance genes in the streptomycetes (bacteria that produce many antibiotics) that have exactly the same biochemical function as the resistance genes” in samples from hospital patients. The similarity (not identical) between the gene sequences suggests that the genes jumped between species, although Davies admits “we can’t yet prove it”. Salyers , involved in the studying of gene jumping suggests that bacteria are tricky when it comes to moving resistant genes.
They can induce other bacteria to initiate genetic swap meets. When one bacterium receives DNA resistance plasmid released by another bacterium, the former releases it’s own plasmid, this is termed as retrotransfer . “This transfer capability gives bacteria the ability to sample DNA from other bacteria,” Salyers says. This is a new form of symbiosis (defined) . “Just about any bacterium can get genes from just about any other bacterium.” Salyers says.
Very distantly related bacteria with resistance genes have been found to have 90 – 95 % similar DNA sequences. This might not qualify for proof but strongly suggests the common origin of genes.
1) Theory : Resistance in Pseudomonas aeruginosa is attributed to Mex efflux pumps.
Method : A combined phenotypic and genotypic approach for the differential diagnosis of r esistance mediated by the transporters MexAB-OprM , MexCD-OprJ , MexEF-OprN , MexXY-OprM was developed. Reference strains harbouring only one specific transporter were used to validate the methodology and its applicability was evaluated towards seven clinical isolates as their resistance mechanisms could not be assigned by the prevalent techniques . MIC measurements with antibiotics [carbenicillin (MexAB-OprM); erythromycin (MexCD-OprJ); norfloxacin and imipenem (MexEF-OprN); gentamicin ( MexXY-OprM)] was used for Phenotypic detection, with and without Phe – Arg – ß – naphthylamide . Semi – quantitative reverse transcription PCR (RT – PCR) for mexC and mexE determined genotypic detection , and by quantitative competitive RT – PCR and real – time PCR for mexA and mexX (correlation between both methods : > 88 % ; overexpression levels – 4.8 – 8.1).
Results: In control strains for all pumps convergence between phenotypic and genotypic methods was observed. Convergence was obtained in 6 of 7 strains for MexXY- OprM and MexEF-OprM , and in 5 of 7 for MexAB-OprM and MexCD-OprJ for clinical isolates.
Conclusions: The data suggests a combination of phenotypic and genotypic approaches in the diagnosis of efflux – mediated resistance in P. aeruginosa.
(Journal of Antimicrobial Chemotherapy 2007 59(3):378-386; doi:10.1093/jac/dkl504)
2) Cross-resistance to fluoroquinolones in multiple-antibiotic-resistant (Mar) Escherichia coli selected by tetracycline or chloramphenicol: decreased drug accumulation associated with membrane changes in addition to OmpF reduction.
S P Cohen, L M McMurry, D C Hooper, J S Wolfson and S B Levy
Department of Molecular Biology and Microbiology, Tufts University School of Medicine, Boston, Massachusetts 02111.
Mar mutants(chromosomal multiple-antibiotic-resistant) of Escherichia coli, cultured on agar having low tetracycline or chloramphenicol contents, were 6- to 18- times less permeable to the fluoroquinolones than E. coli K-12 or E. coli C parental strains. The emergence frequency of these mutants was at least 1,000 times higher than that of those selected by the fluoroquinolone norfloxacin directly. When Mar mutants(not wild-type) , were coated on norfloxacin, mutants resistant to high levels of norfloxacin (2 micrograms/ml) appeared at a comparatively higher (approximately 10(-7] frequency. In addition to decreased amounts of OmpF, Mar mutants had also changes in outer membrane protein and were four- to eight times less susceptible to fluoroquinolones than was an ompF::Tn5 mutant lacking only OmpF. Conglomeration of [3H]norfloxacin was more than three times lower in the Mar mutants than in wild-type and two times lower than in the OmpF-deficient derivative. These differences were not attributed to a change in the endogenous active efflux system for norfloxacin in E. coli. Norfloxacin-induced inhibition of DNA synthesis was three times lower in intact cells of a Mar mutant than in susceptible cells, but this difference was not there in toluene-permeabilized cells.
Insertion of Tn5 into marA (min 34.05 on the chromosome) led to a return of the wild-type patterns of norfloxacin accumulation, fluoroquinolone and other antimicrobial agent susceptibilities, and outer membrane protein profile, including partial restoration of OmpF.
Conclusion : These findings strongly suggest that marA-dependent fluoroquinolone resistance is linked to decreased cell permeability . Once mutated to marA, cells can achieve high levels of quinolone resistance at a relatively high frequency. (Antimicrob Agents Chemother. 1989 August; 33(8): 1318-1325)
Way Forward :-
The synthesis of large numbers of antibiotics over the past three decades has caused today’s crisis . Bugs have learnt to defy their killers as a result of chromosomal change . The most rational thing to do would be to use antibiotics more judiciously and let the pace of the modern scenario slow down while a medical miracle steps in to defy the notorious bugs .
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