Combined Use of the Modified Hodge Test and Carbapenemase Inhibition Test for Detection of Carbapenemase-Producing Enterobacteriaceae and Metallo-beta-Lactamase-Producing Pseudomonas spp

BACKGROUND: We evaluated the combined use of the modified Hodge test (MHT) and carbapenemase inhibition test (CIT) using phenylboronic acid (PBA) and EDTA to detect carbapenemase-producing Enterobacteriaceae (CPE) and metallo-beta-lactamase (MBL)-producing Pseudomonas spp. METHODS: A total of 49 isolates of CPE (15 Klebsiella pneumoniae carbapenemase [KPC], 5 Guiana extended-spectrum beta-lactamase [GES]-5, 9 New Delhi metallo-beta-lactamase [NDM]-1, 5 Verona integron-encoded metallo-beta-lactamase [VIM]-2, 3 imipenem-hydrolyzing beta-lactamase [IMP], and 12 oxacillinase [OXA]-48-like), 25 isolates of MBL-producing Pseudomonas spp. (14 VIM-2 and 11 IMP), and 35 carbapenemase-negative controls were included. The MHT was performed for all isolates as recommended by the Clinical and Laboratory Standards Institute. Enhanced growth of the indicator strain was measured in mm with a ruler. The CIT was performed by directly dripping PBA and EDTA solutions onto carbapenem disks that were placed on Mueller-Hinton agar plates seeded with the test strain. RESULTS: Considering the results of the MHT with the ertapenem disk in Enterobacteriaceae and Pseudomonas spp., the CIT with the meropenem disk in Enterobacteriaceae, and the imipenem disk in Pseudomonas spp., three combined disk tests, namely MHT-positive plus PBA-positive, EDTA-positive, and MHT-positive plus PBA-negative plus EDTA-negative, had excellent sensitivity and specificity for the detection of KPC- (100% sensitivity and 100% specificity), MBL- (94% sensitivity and 100% specificity), and OXA-48-like-producing isolates (100% sensitivity and 100% specificity), respectively. CONCLUSIONS: Combined use of the MHT and CIT with PBA and EDTA, for the detection of CPE and MBL-producing Pseudomonas spp., is effective in detecting and characterizing carbapenemases in routine laboratories.

Suppression of Eis and expression of Wag31 and GroES in Mycobacterium tuberculosis cytosol under anaerobic culture conditions.

A major impediment in chemotherapy of Tuberculosis (TB) is the persistence of M. tuberculosis in a latent or dormant state, possibly perpetuated by paucity of oxygen within the lung granuloma. Proteome analysis of the anaerobically persisting microbe could therefore provide novel targets for drugs against latent TB infection (LTBI). An Indian clinical isolate of M. tuberculosis was cultured under aerobic and anaerobic conditions following Wayne’s hypoxia model and its cytosolic proteins were resolved by two-dimensional gel electrophoresis (2DE). Peptide mass fingerprinting of 32 differentially expressed spots using MALDI TOF-TOF MS-MS resulted in identification of 23 proteins. Under the anaerobic culture conditions, expression of 12 of these proteins was highly suppressed (>2 fold reduction in spot volumes), with 4 of them (GrpE, CanB, MoxR1 and Eis) appearing as completely suppressed since corresponding spots were not detectable in the anaerobic sample. On the other hand, 4 proteins were highly expressed, with two of them (Wag31 and GroES) being uniquely expressed under anaerobic conditions. Suppression of Eis could make the anaerobically persisting bacilli susceptible to the aminoglycoside antibiotics which are known to be acetylated and inactivated by Eis. Although all 4 over-expressed proteins can be considered as putative drug targets for LTBI, Wag31 appears particularly interesting in view of its role in the cell wall biogenesis.

Actividad inhibitoria de dihidroxifenil propenona sobre betalactamasas de Enterobacter cloacae: estudio preliminar en el desarrollo de fármacos para enfrentar la resistencia bacteriana / Inhibitory activity of dihydroxy-phenyl-propenone on betalactamase of Enterobacter cloacae : Preliminary study for drug development to overcome bacterial resistance

Introducción . Los microorganismos patógenos como Enterobacter cloacae producen betalactamasas que les confieren resistencia frente a los antibióticos betalactámicos; se ha identificado, además, la actividad limitada de los inhibidores enzimáticos, de modo que la única posibilidad de enfrentar la resistencia es el diseño de nuevos fármacos y su uso racional. Objetivo. Evaluar el efecto de la chalcona dihidroxifenil propenona sobre un aislamiento clínico de E. cloacae y sobre la betalactamasa aislada a partir de este microorganismo resistente como un aporte en la búsqueda de compuestos inhibidores de las betalactamasas. Materiales y métodos. Se sintetizó la chalcona dihidroxifenil propenona y se evaluó su efecto sobre el aislamiento clínico de E. cloacae para determinar la concentración inhibitoria mínima mediante el método de microdilución en caldo y con la betalactamasa purificada mediante cromatografía de afinidad se realizaron estudios espectrofotométricos de cinética enzimática. Resultados. La concentración inhibitoria mínima de la dihidroxifenil propenona sobre E. cloacae fue de 35 µg/ml; el porcentaje de recuperación de la betalactamasa a partir del microorganismo fue de 31,75 %; en el estudio cinético se evidenció actividad inhibitoria de acuerdo con los parámetros cinéticos de V max =1,7 x 10 -3 µM/minuto y K M´ =2330 µM. Conclusión. La chalcona dihidroxifenil propenona ejerce su actividad inhibitoria por medio de la interacción con la betalactamasa y, de esta manera, protege la integridad estructural de los antibióticos betalactámicos; dicho efecto sinérgico la convierte en un compuesto promisorio en la búsqueda de alternativas para enfrentar la resistencia bacteriana.

Introduction: Enterobacter cloacae is a pathogenic microorganism with the ability to produce betalactamase enzymes, which makes them resistant to betalactamic antibiotics. Additionally, the limited activity of enzymatic inhibitors has been identified, and, therefore, the design of new drugs and the promotion of their rational use are the only possibilities to overcome this problem. Objective: The aim of this research was to evaluate the effect of dihydroxy-phenyl-propenone on a clinical isolate of E. cloacae , as well as its activity on a betalactamase isolated from this resistant microorganism in order to contribute to the search for new betalactamase inhibitors. Materials and methods: Dihydroxy-phenyl-propenone chalcone was synthesized and evaluated on a clinical isolate of E. cloacae to determine the minimum inhibitory concentration by broth microdilution; once the betalactamase enzyme was purified by affinity chromatography, a spectrophotometric analysis was done to evaluate its kinetic activity. Results: The minimum inhibitory concentration value of dihydroxy-phenyl-propenone on E. cloacae was 35 µg/ml; the recovery percentage of the betalactamase from the microorganism was 31.75% and the kinetic parameters were V max =1.7 x 10 -3 µM/min and K M = 2330 µM, which show an important inhibitory activity. Conclusion: Dihydroxy-phenyl-propenone has shown inhibitory activity on betalactamase enzymes and the ability to protect the chemical integrity of betalactamic antibiotics; this synergistic effect turns it into a promising compound in the search for new alternatives to overcome bacterial resistance.

Correlation of Ciprofloxacin Resistance with the AdeABC Efflux System in Acinetobacter baumannii Clinical Isolates

BACKGROUND: Acinetobacter baumannii is one of the most important pathogens capable of colonization in burn patients, leading to drug-resistant wound infections. This study evaluated the distribution of the AdeABC efflux system genes and their relationship to ciprofloxacin resistance in A. baumannii isolates collected from burn patients. METHODS: A total of 68 A. baumannii clinical strains were isolated from patients hospitalized in Motahari Burns Center in Tehran, Iran. Ciprofloxacin susceptibility was tested by the disk diffusion and agar dilution methods. PCR amplification of the adeRS-adeB drug efflux genes was performed for all resistant and susceptible isolates. To assess the role of the drug efflux pump in ciprofloxacin susceptibility, carbonyl cyanide 3-chlorophenylhydrazone (CCCP) was used as an efflux pump inhibitor (EPI). RESULTS: Approximately 95.6% of the Acinetobacter isolates were resistant to ciprofloxacin, with minimum inhibitory concentration (MIC) values ranging from 4 to > or =128 microg/mL. The susceptibility of 86.1% of the resistant isolates increased by factors of 2 to 64 in the presence of CCCP. All resistant isolates were positive for the adeRS-adeB genes, and 73.2% of them had mutations in the AdeRS regulatory system. CONCLUSIONS: The results showed that AdeABC genes are common in A. baumannii, which might be associated with ciprofloxacin non-susceptibility, as indicated by the observed linkage to the presence of the genes essential for the activity of the AdeABC, several single mutations occurring in the adeRS regulatory system, and an increase of ciprofloxacin susceptibility in the presence of a CCCP EPI.

Actinobacteriological research in India.

Actinobacteria are important sources of compounds for drug discovery and have attracted considerable pharmaceutical, chemical, agricultural and industrial interests. Actinobacteriological research is still in its infancy in India. Early work on actinobacteria started in the 20th century and mostly focused on studying the diversity, identification and screening for antibiotics, enzymes and enzyme inhibitors. Exploration of diverse habitats for the isolation of actinobacteria, have yielded till date 23 novel species. Screening of actinobacteria for antagonistic activity, has led to the discovery of four novel antibiotics. Research on enzymes mostly covered lipases, amylases, proteases, endoglucanases, α-galactosidases, pectin lyases, xylanases, L-asparaginases, L-glutaminase and cellulases. Research on exploiting actinobacteria for other purposes such as production of enzyme inhibitors, single cell protein, bioemulsifier and biosurfactants is still in the experimental stage. This review compiles the work done in last few years, with an emphasis on actinobacterial diversity and bioprospecting for pharmaceutically important compounds like antibiotics, enzymes and other important applications. The chemical creativity and biotechnological potential of Indian actinobacterial strains are yet to be fully explored. A national strategy is required consistent with the opportunities provided by CBD-Nagoya protocol.

Effect of efflux pump inhibitors on drug susceptibility of ofloxacin resistant Mycobacterium tuberculosis isolates.

Background & objectives In drug resistant, especially multi-drug resistant (MDR) tuberculosis, fluoroquinolones (FQs) are used as second line drugs. However, the incidence of FQ-resistant Mycobacterium tuberculosis is rapidly increasing which may be due to extensive use of FQs in the treatment of various other diseases. The most important known mechanism i.e., gyrA mutation in FQ resistance is not observed in a significant proportion of FQ resistant M. tuberculosis isolates suggesting that the resistance may be because of other mechanisms such as an active drug efflux pump. In this study we evaluated the role of the efflux pumps in quinolone resistance by using various inhibitors such as carbonyl cyanide m-chlorophenyl hydrazone (CCCP), 2,4-dinitrophenol (DNP) and verapamil, in clinical isolates of M. tuberculosis. Methods A total of 55 M. tuberculosis clinical isolates [45 ofloxacin (OFL) resistant and 10 ofloxacin sensitive] were tested by Resazurin microtitre assay (REMA) to observe the changes in ofloxacin minimum inhibitory concentration (MIC) levels in presence of efflux inhibitors as compared to control (without efflux inhibitor). Results The MIC levels of OFL showed 2-8 folds reduction in presence of CCCP (16/45; 35.5%), verapamil (24/45; 53.3%) and DNP (21/45; 46.6%) while in case of isolates identified as OFL sensitive these did not show any effect on ofloxacin MICs. In 11 of 45 (24.5%) isolates change in MIC levels was observed with all the three inhibitors. Overall 30 (66.6%) isolates had reduction in OFL MIC after treatment with these inhibitors. A total of eight isolates were sequenced for gyrA gene, of which, seven (87.5%) showed known mutations. Of the eight sequenced isolates, seven (87.5%) showed 2 to 8 fold change in MIC in presence of efflux inhibitors. Interpretation & conclusions Our findings suggest the involvement of active efflux pumps of both Major Facilitator Super Family (MFS) family (inhibited by CCCP and DNP) and ATP Binding Cassette (ABC) transporters (inhibited by verapamil) in the development of OFL resistance in M. tuberculosis isolates. Epidemiological significance of these findings needs to be determined in prospective studies with appropriate number of samples / isolates.

Isolation and characterization of a metal ion-dependent alkaline protease from a halotolerant Bacillus aquimaris VITP4.

A halotolerant bacterium Bacillus acquimaris VITP4 was used for the production of extracellular protease. Fractional precipitation using ammonium chloride was used to obtain the enzyme. The protease exhibited optimum activity at pH 8.0 and 40°C and retained 50% of its optimal proteolytic activity even in the presence of 4 M NaCl, suggesting that it is halotolerant. The molecular mass of protease, as revealed by SDS-PAGE was found to be 34 kDa and the homogeneity of the enzyme was confirmed by gelatin zymography and reverse-phase HPLC. Upon purification, the specific activity of th enzyme increased from 533 U/mg to 1719 U/mg. Protease inhibitors like phenyl methane sulphonyl fluoride and 2-mercaptoethanol did not affect the activity of the enzyme, but EDTA inhibited the activity, indicating the requirement of metal ions for activity. Cu­­­2+, Ni2+ and Mn2+ enhanced the enzyme activity, but Zn2+, Hg2+ and Fe2+ decreased the activity, while Mg2+, Ca2+ and K+ had no effect on the enzyme activity. The protease was quite stable in the presence of cationic (CTAB), anionic (SDS) and neutral detergents (Triton X-100 and Tween-20) and exhibited antimicrobial activity against selected bacterial and fungal strains. The stability characteristics and broad spectrum antimicrobial activity indicated the potential use of this protease in industrial applications.

Isolation of a Klebsiella pneumoniae Isolate of Sequence Type 258 Producing KPC-2 Carbapenemase in Korea / 대한진단검사의학회지

Carbapenem-resistant Klebsiella pneumoniae isolates producing K. pneumoniae carbapenemases (KPC) were first reported in the USA in 2001, and since then, this infection has been reported in Europe, Israel, South America, and China. In Korea, the first KPC-2-producing K. pneumoniae sequence type (ST) 11 strain was detected in 2010. We report the case of a patient with a urinary tract infection caused by KPC-2-producing K. pneumoniae. This is the second report of a KPC-2-producing K. pneumoniae infection in Korea, but the multilocus sequence type was ST258. The KPC-2-producing isolate was resistant to all tested beta-lactams (including imipenem and meropenem), amikacin, tobramycin, ciprofloxacin, levofloxacin, and trimethoprim-sulfamethoxazole, but was susceptible to gentamicin, colistin, polymyxin B, and tigecycline. The KPC-2-producing isolate was negative to phenotypic extended-spectrum beta-lactamase (ESBL) and AmpC detection tests and positive to modified Hodge test and carbapenemase inhibition test with aminophenylboronic acid.

Inhibición de la actividad proteolítica de Helicobacter pylori por suero inmune de ratón / Inhibition of the proteolytic activity of Helicobacter pylori by mouse immune serum

The proteolytic activity (PA) of some microorganisms is an important pathogenic factor during tissue invasion. However, its role in Helicobacter pylori infection is not clear. Due to the importance of the immunological response to inhibit pathogenic factors of microorganisms, this study aims to establish an in vitro system to detect inhibitory antibodies to the PA of H. pylori in mouse serum. We obtained mouse sera from animals immunized by oral and intraperitoneal inoculations with the raw bacterial extract (BE) of H. pylori, in which we had previously detected PA. The degradation of azocasein subtract for BE was inhibited in 49.23 and 22.6 using 5 micrograms/ml of serum proteins (SP) from oral and intraperitoneal inoculation, respectively. However, when using more than 25 micrograms/ml of SP of immune serum, PA was inhibited in a similar way than with control serum. In conclusion we present a methodology for the detection of inhibitory antibodies to PA of H. pylori in the serum of the immunized mouse.

Inhibitor to the cell-bound haemagglutinin of Vibrio cholerae.

An inhibitor to cell-bound HA was found to be produced at the non-haemagglutinating phase of the culture cycle by a classical vibrio strain which produced a cell-bound HA early and transiently during its growth. The HA-negative filtrate obtained from the late log-culture was found to inhibit the cell-bound HA activity produced by the same vibrio strain. It was also found to be produced early in shaking cultures at 37 degrees C and to mask the activity of early cell-bound HA in whole culture tests. This inhibitor is suggested to be responsible for the failure to obtain HA activity or adhesive vibrio cells grown at 37 degrees C and for the transient expression of cell-bound HA by some V. cholerae strains.