Quorum-sensing system in Acinetobacter baumannii: a potential target for new drug development.

Acinetobacter baumannii causes several nosocomial infections and poses major threat when it is multidrug resistant. Even pan drug-resistant strains have been reported in some countries. The intensive care unit (ICU) mortality rate ranged from 45.6% to 60.9% and it is as high as 84.3% when ventilator-associated pneumonia was caused by XDR (extensively drug resistant) A. baumannii. Acinetobacter baumannii constituted 9.4% of all Gram-negative organisms throughout the hospital and 22.6% in the ICUs according to a study carried out in an Indian hospital. One of the major factors contributing to drug resistance in A. baumannii infections is biofilm development. Quorum sensing (QS) facilitates biofilm formation and therefore the search for 'quorum quenchers' has increased recently. Such compounds are expected to inhibit biofilm formation and hence reduce/prevent development of drug resistance in the bacteria. Some of these compounds also target synthesis of some virulence factors (VF). Several candidate drugs have been identified and are at various stages of drug development. Since quorum quenching, inhibition of biofilm formation and inhibition of VF synthesis do not pose any threat to the DNA replication and cell division of the bacteria, chances of resistance development to such compounds is presumably rare. Thus, these compounds ideally qualify as adjunct therapeutics and could be administered along with an antibiotic to reduce chances of resistance development and also to increase the effectiveness of antimicrobial therapy. This review describes the state-of-art in QS process in Gram-negative bacteria in general and in A. baumannii in particular. This article elaborates the nature of QS mediators, their characteristics, and the methods for their detection and quantification. Various potential sites in the QS pathway have been highlighted as drug targets and the candidate quorum quenchers which inhibit the mediator's synthesis or function are enlisted.

A structural, epidemiological & genetic overview of Klebsiella pneumoniae carbapenemases (KPCs).

Klebsiella pneumoniae carbapenemases (KPCs) are plasmid encoded carbapenem hydrolyzing enzymes which have the potential to spread widely through gene transfer. The instability of upstream region of blaKPC accelerates emergence of different isoforms. Routine antibiotic susceptibility testing failed to detect KPC producers and some commercial kits have been launched for early identification of KPC producers. Notable among the drugs under development against KPC are mostly derivatives of polymixin; ß-lactamase inhibitor NXL104 with combination of oxyimino cephalosporin as well as with ceftazidime; a novel tricyclic carbapenem, LK-157, potentially useful against class A and class C enzymes; BLI-489-a bicyclic penem derivative; PTK-0796, a tetracycline derivative and ACHN-490. Combination therapy might be preferable to control KPC infections in immediate future. Clinicians are likely to opt for unconventional combinations of antibiotics to treat KPC infections because of unavailability of alternative agents. The KPCs have become endemic in many countries but there is no optimal treatment recommendation available for bacteria expressing KPCs. Reports of outbreaks involving KPCs have focused mainly on laboratory identification, empirical treatment outcomes and molecular epidemiology. This review includes information on the emergence of KPC variants, limitations of phenotyping methods, available molecular methods for identification of the KPC variants and treatment options highlighting the drugs under development.

Biomarkers for the management of pre-eclampsia in pregnant women.

Pre-eclampsia (PE) is a pregnancy related disorder characterized by hypertension and proteinuria noticeable after 20 wk of gestation. It is a leading cause of maternal and foetal mortality and morbidity worldwide. The aetiology of the disease is unknown, but recent studies have revealed that this disorder appears to originate in placenta and is characterized by widespread maternal endothelial dysfunction. Till date, delivery of placenta is the only cure for the disease. So, there is a need for the identification of highly specific and sensitive biochemical markers that would allow early identification of patients at risk and thus help in providing proper prenatal care. Several promising biomarkers have been proposed, alone or in combination, that may help in predicting women who are likely to develop PE. Maternal serum concentrations of these biomarkers either increase or decrease in PE during gestation. This review focuses on the various biomarkers available and their utility in predicting pre-eclampsia.

Microbial pathogenesis: an insight into Mycobacterium tuberculosis.

Tuberculosis, as yet, is far from being controlled. Several reasons can be attributed to this, a major contributing factor being the development of resistance to the currently available drugs due to the successful adaptation of the pathogen. Most of the inferences about the pathogen are based on the observation of mycobacteria grown in synthetic media in vitro and of the mycobacteria maintained in macrophages simulating the in vivo conditions. Molecular studies in mycobacteria had been slow to come due to the difficulty in the generation of mutants. However, new technologies that have now been developed for studying in vivo expressed molecules in other bacterial systems are being successfully applied to mycobacteria, especially the pathogenic M. tuberculosis. Additionally, an equally important factor in the study of the disease is the genetic predisposition of population to the infection. New findings link the Nramp1 and Toll receptor polymorphisms to susceptibility to infectious diseases.

Direct diagnosis ofMycobacterium tuberculosis in blood samples of HIV infected patients by polymerase chain reaction.

We have developed a simple, economical and reproducible method for processing blood samples from HIV infected patients for diagnosis of tuberculosis. The procedure was validated on 55 samples selected for tuberculosis based on clinical criteria. 52 patients had radiological changes indicative of pulmonary tuberculosis of which only 28 were positive for AFB in sputum (sensitivity 54%) and 27 for tuberculin (sensitivity 52%). 26 HIV positive patients who showed positive X-ray did not react to tuberculin. The genus PCR probe missed 3 samples (sensitivity 94%) compared to X-ray.M.tuberculosis was detected in the blood of all X-ray positive cases by PCR using TB400 probe (sensitivity 100%) and another probe forM. tuberculosis, IS6110, missed 6 of them (sensitivity 88% compared to X-ray and 89% compared to TB400). It is proposed that this simple sample processing method could be used to screen all blood samples quickly for mycobacteremia using the genus PCR and only those positive for mycobacteria need to be tested forM.tuberculosis. This would save the scarce resources and time by reducing significantly the number of samples to be screened for species confirmation.

Isolation, PCR based identification, and sensitivity pattern of environmental mycobacteria from leprosy and tuberculosis patients.

We have isolated and identified the biotype of environmental mycobacteria from the expectorate of leprosy patients, their contacts, their drinking water supply and also from the sputa samples of tuberculosis patients. 78% of the isolates from lepromatous leprosy patients and their contacts wereMycobacterium fortuitum- chelonae complex (MFC), 9%Mycobacterium avium complex (MAC), 9%Mycobacterium scrofulaceum and 4% wereMycobacterium smegmatis. Among the isolates from tuberculosis patients 63% belonged toM. fortuitum- chelonae complex, 19% toM. avium complex, 12% toMycobacterium Kansasii and 6% toM. smegmatis. All the isolates were multi-drug resistant when tested for sensitivity total of 21 drugs. TheMycobacterium fortuitum-chelonae complex organisms from leprosy contacts were more sensitive to rifampicin than those isolated from lepromatous leprosy and tuberculosis patients. Among 23 isolates from leprosy patients one isolate was resistant to 20 drugs, one isolate to 17 drugs and another isolate was resistant to 13 drugs. Among the 18 isolates from drinking water supply six showed resistance to more than 12 drugs. Polymerase Chain Reaction (PCR) and subsequent hybridisation with specific probes confirmed all the isolated strains as nontuberculous mycobacteria (Using genus primers and probe sensitivity 100%) and none asM. tuberculosis, suggesting that PCR could be used to rapidly identify mycobacteria at the genus level and to rule out tuberculosis in leprosy patients at an early stage to decide on appropriate course of therapy.

Polymerase chain reaction in the diagnosis of tuberculosis.

A rapid, sensitive, specific and yet economical method for the diagnosis ofM. tuberculosis and other mycobacteria in clinical specimen is a desperate and urgent requirement of the day in the laboratory diagnosis and hence management of tuberculosis. This need is further accentuated by emerging diseases like multi drug resistant tuberculosis, tuberculosis in AIDS patients and opportunistic mycobacterial infections, which do not respond to conventional anti TB therapy. Molecular methods, particularly PCR based detection ofM. tuberculosis, has come a long way since it was first described about fifteen years ago. Several probes have been developed and some of them, particularly the IS6110 and TB400 have been validated on several clinical samples. The latter has been validated on a variety of clinical specimens along with a simple sample processing method. Polymerase chain reaction based diagnosis ofM. tuberculosis has been introduced as one of the routine/confirmatory tests in clinical microbiology laboratory in some countries like Canada, the United States and the United Kingdom several years ago. The possibility of introducing PCR based direct diagnosis of drug resistance is being explored in some laboratories, particularly for drugs like rifampicin. The evolution and application of PCR for diagnosis ofM. tuberculosis is being analysed and discussed in this review.

Polymerase chain reaction and non-radioactive gene probe based identification of mosquito larvicidal strains of Bacillus sphaericus and monitoring of B. sphaericus 1593M, released in the environment.

In attempts towards an accurate monitoring in the environment, the status of the deliberate release of Bacillus sphaericus, a powerful mosquito larvicidal agent, as well as to evolve a rapid method of screening for potent isolates of B. sphaericus, we have developed a polymerase chain reaction (PCR) method of analysis. Using specific primers spanning the 5' and 3' ends of the coding sequences of these two mosquito larvicidal genes, a 1.3 and a 1.1 kb product from gene A and a 2.6 kb product from gene B have been amplified by PCR. The primers and the products amplified from them in PCR are highly specific for B. sphaericus. We used digoxigenin based non-radioactive chemiluminescence method for the detection of PCR products and the sensitivity of the method was high enough to detect the presence of 1 to 5 cells of B. sphaericus. A simple and inexpensive sample processing procedure has also been developed for direct PCR amplification of B. sphaericus DNA from field samples collected from areas where it had been applied. Several highly toxic to non-toxic strains of B. sphaericus were screened with these primers by PCR for the presence of either or both of these toxin genes. The results indicate that there is a good correlation between the presence of both genes with higher toxicity of the strains.

Specificity of diagnostic PCR amplification for M. avium using the probe pMAV22.

We characterized the serovar specificity of the probe pMAV22 using ATCC isolates. Primers from this sequence amplified DNA from serovars 1-6, 8-10, and ATCC strain 19075 but did not amplify MAIS serovars 7, 11, 12, 14, 17-20. This confirmed that the M. avium probe pMAV22 is specific for M. avium, not M. intracellulare.

Comparison of genus- and species-specific probes for PCR detection of mycobacterial infections.

We describe experiments comparing use of different DNA probes to detect mycobacteria in clinical specimens after PCR. The objective was to assess correlation between results using Mycobacterium genus-specific, and species-specific M. tuberculosis probes. Given sufficient concordance, sequential use of such probes would provide a useful screening tool. An evaluation of genus-specific probes compared use of repetitive sequences in the clone pMAv17 with the 65-kDa sequences. Sensitivity was 100% for pMAv17, 93% using the 65-kDa sequence; specificity was 70% for both. We then compared M. tuberculosis-specific probes developed by us (Tb400) with IS6110 and mpt40. Sensitivity using Tb400 was 100%; using IS6110 was 97%, and using mpt40 was 50%. Specificity using Tb400 and IS6110 was 68%, and was 70% using mpt40. Fourteen specimens which were PCR-positive and culture-negative, were positive using both genus probes, and the M. tuberculosis-specific probes Tb400, and IS6110. Ten of these were positive using mpt40.

Diagnosis of Mycobacterium avium bacteremia by polymerase chain reaction.

We describe a rapid polymerase chain reaction (PCR)-based test for diagnosing Mycobacterium avium directly from blood specimens. Blood was collected in anticoagulant (EDTA) from patients who also had blood cultures performed by the lysis-centrifugation method. Blood samples were centrifuged on a Ficoll-Hypaque gradient to purify peripheral blood mononuclear cells. The purified cells were washed and incubated in the presence of Chelex-100 (a divalent cation-binding resin), boiled to release mycobacterial DNA, and then amplified with M. avium-specific PCR primers. Amplification was detected by hybridization with radiolabelled probe, and the results were compared with the culture results. The PCR assay gave positive results for 12 of 15 specimens that were taken from patients with positive cultures for M. avium complex (sensitivity, 80%). The three PCR-negative specimens in this group showed evidence of PCR inhibition. The PCR assay gave positive results for 32 of 228 specimens taken from patients with negative cultures (specificity, 86%). Of these 32 PCR-positive culture-negative specimens, 27 were also positive when amplified with primers specific for the genus Mycobacterium, suggesting that PCR may be more sensitive than culture.

A simple method for diagnosing M. tuberculosis infection in clinical samples using PCR.

Species identification of Mycobacterium tuberculosis remains a cumbersome process. We have developed a simple method for treating clinical samples which permits direct polymerase chain reaction (PCR) amplification of mycobacterial target DNA without organic extraction. Samples were boiled for 30 min in TE-Triton, then were subjected to 30 cycles of amplification using primers derived from the repetitive clone pMTb4 developed by Patel and coworkers (1990; Journal of Clinical Microbiology 28, 513). Specificity of amplification was confirmed by hybridization with a specific probe labelled non-isotopically. In a model system consisting of cultured bacilli, this system routinely allows detection of a single organism in a sample. In preliminary studies examining applicability of this method to 96 clinical samples, 74 were positive by both PCR and mycobacterial culture, and eight were negative by both methods. Fourteen samples were negative by culture and positive by PCR, and none were positive by culture and negative by PCR. These results suggest that the PCR method may provide a sensitive alternative to conventional species-specific diagnostic methods, and that non-isotopic labelling can be used to detect hybridization in this assay.

Aspartate metabolism in Mycobacterium avium grown in host tissue and axenically and in Mycobacterium leprae.

Aspartokinase activity was detected in extracts from Mycobacterium leprae (recovered from armadillo liver) and in Mycobacterium avium grown axenically and in vivo. Homoserine dehydrogenase activity was only detected in M. leprae and in M. avium grown axenically. Activities, when detected, were 50 to 70% lower in M. leprae or M. avium grown in vivo than in axenically grown M. avium. In these two pathogenic mycobacteria, aspartokinase and homoserine dehydrogenase are subject to feedback inhibition by methionine - an additional regulator over those observed for the enzymes from Mycobacterium smegmatis. Intact mycobacterium incorporated carbon from [U-14C]aspartate into the aspartate family of amino acids (threonine, isoleucine, methionine and lysine) though the rate of incorporation in M. avium grown in vivo was about half that in M. avium grown axenically.

Metabolism of aspartate in Mycobacterium smegmatis.

Mycobacterium smegmatis grows best on L-asparagine as a sole nitrogen source; this was confirmed. [14C]Aspartate was taken up rapidly (46 nmol.mg dry cells-1.h-1 from 1 mM L-asparagine) and metabolised to CO2 as well as to amino acids synthesised through the aspartate pathway. Proportionately more radioactivity appeared in the amino acids in bacteria grown in medium containing low nitrogen. Activities of aspartokinase and homoserine dehydrogenase, the initial enzymes of the aspartate pathway, were carried by separate proteins. Aspartokinase was purified as three isoenzymes and represented up to 8% of the soluble protein of M. smegmatis. All three isoenzymes contained molecular mass subunits of 50 kDa and 11 kDa which showed no activity individually; full enzyme activity was recovered on pooling the subunits. Km values for aspartate were: aspartokinases I and III, 2.4 mM; aspartokinase II, 6.4 mM. Aspartokinase I was inhibited by threonine and homoserine and aspartokinase III by lysine, but aspartokinase II was not inhibited by any amino acids. Aspartokinase activity was repressed by methionine and lysine with a small residue of activity attributable to unrepressed aspartokinase I. Homoserine dehydrogenase activity was 96% inhibited by 2 mM threonine; isoleucine, cysteine and valine had lesser effects and in combination gave additive inhibition. Homoserine dehydrogenase was repressed by threonine and leucine. Only amino acids synthesised through the aspartate pathway were tested for inhibition and repression. Of these, only one, meso-diaminopimilate, had no discernable effect on either enzyme activity.

Effect of homoserine on growth of Mycobacterium smegmatis: inhibition of glutamate transport by homoserine.

Homoserine strongly inhibited growth of Mycobacterium smegmatis in medium containing glutamate as the sole source of nitrogen but was without effect when asparagine, alanine or glutamine was the sole nitrogen source. It was readily taken up by glutamate-grown cells, reaching an intracellular concentration of over 20 mM after 4 h incubation. The primary site of action of homoserine was deduced to be the non-competitive inhibition of glutamate transport.