Comparative testing of disinfectant efficacy on planktonic bacteria and bacterial biofilms using a new assay based on kinetic analysis of metabolic activity.

AIMS: The aim of our study was to develop a new reproducible method for disinfectant efficacy testing on bacterial biofilms and to evaluate the efficacy of different disinfectants against biofilms. Clinical multidrug-resistant strains were chosen as test isolates to ensure practical relevance. METHODS AND RESULTS: We compared the standard qualitative suspension assay for disinfectant testing, which does not take into account biofilm formation, to the new biofilm viability assay that uses kinetic analysis of metabolic activity in biofilms after disinfectant exposure to evaluate disinfectant efficacy. In addition, the efficacy of four standard disinfectants to clinical isolates was tested using both methods. All tested disinfectants were effective against test isolates when in planktonic state using the standard qualitative suspension assay, while disinfectants were only weakly effective against bacteria in biofilms. CONCLUSIONS: Disinfectant efficacy testing on planktonic organisms ignores biofilms and overestimates disinfectant susceptibility of bacteria. However, biofilm forming, e.g. on medical devices or hospital surfaces, is the natural state of bacterial living and needs to be considered in disinfectant testing. SIGNIFICANCE AND IMPACT OF THE STUDY: Although bacterial biofilms are the predominant manner of bacterial colonization, most standard procedures for antimicrobial susceptibility testing and efficacy testing of disinfectants are adapted for application to planktonic bacteria. To our knowledge, this is the first study to use a newly developed microplate-based biofilm test system that uses kinetic analysis of the metabolic activity in biofilms, after disinfectant exposure, to evaluate disinfectant efficacy. Our study shows that findings obtained from disinfectant efficacy testing on planktonic bacteria cannot be extrapolated to predict disinfectant efficacy on bacterial biofilms of clinically relevant multidrug-resistant organisms.

Determinants for persistence of Pseudomonas aeruginosa in hospitals: interplay between resistance, virulence and biofilm formation.

Pseudomonas aeruginosa (Pa) is one of the major bacterial pathogens causing nosocomial infections. During the past few decades, multidrug-resistant (MDR) and extensively drug-resistant (XDR) lineages of Pa have emerged in hospital settings with increasing numbers. However, it remains unclear which determinants of Pa facilitated this spread. A total of 211 clinical XDR and 38 susceptible clinical Pa isolates (nonXDR), as well as 47 environmental isolates (EI), were collected at the Heidelberg University Hospital. We used RAPD PCR to identify genetic clusters. Carriage of carbapenamases (CPM) and virulence genes were analyzed by PCR, biofilm formation capacity was assessed, in vitro fitness was evaluated using competitive growth assays, and interaction with the host's immune system was analyzed using serum killing and neutrophil killing assays. XDR isolates showed significantly elevated biofilm formation (p < 0.05) and higher competitive fitness compared to nonXDR and EI isolates. Thirty percent (62/205) of the XDR isolates carried a CPM. Similarities in distribution of virulence factors, as well as biofilm formation properties, between CPM+ Pa isolates and EI and between CPM- and nonXDR isolates were detected. Molecular typing revealed two distinct genetic clusters within the XDR population, which were characterized by even higher biofilm formation. In contrast, XDR isolates were more susceptible to the immune response than nonXDR isolates. Our study provides evidence that the ability to form biofilms is an outstanding determinant for persistence and endemic spread of Pa in the hospital setting.

Susceptibility of multidrug resistant clinical pathogens to a chlorhexidine formulation.

Multidrug resistant pathogens are a widespread problem in the hospital setting especially on intensive care units (ICU). This study evaluated the susceptibility of clinical isolates of gramnegative extensively drug resistant organisms (XDR), methicillinresistant Staphylococcus aureus (MRSA), and vancomycin-resistant Enterococcus (VRE) to a proprietary chlorhexidine digluconate (CHG) formulation used in one brand of CHG-impregnated cloths. Ten isolates each of XDR Pseudomonas aeruginosa, XDR Acinetobacter baumannii, XDR Klebsiella pneumoniae, XDR Escherichia coli, MRSA, and vancomycin-resistant Enterococcus faecium from our hospital were tested. All isolates were susceptible to the proprietary CHG formulation (0.5%, 1%, 2%), with 99% to 100% suppression of growth at the earliest time point in time kill assays (1 minute for gram-positive and 15 seconds for gram-negative organisms). Minimum inhibitory concentrations ranged from 1 : 4096 to 1 : 65536 for MRSA, 1 : 1024 to 1 : 2048 for VRE, 1 : 2048 to 1 : 4096 for XDR E. coli, 1 : 512 to 1 : 2048 for XDR A. baumannii, 1 : 512 to 1 : 1024 for XDR P. aeruginosa, and 1 : 512 to 1 : 1024 for XDR K. pneumoniae. Cloths impregnated with this CHG formulation provide effective protection against colonization and infection by many pathogens. This study provides in vitro evidence that the proprietary CHG formulation used in one brand of CHG-impregnated cloths is effective against XDR gram-negative organisms, MRSA, and VRE.

Comparison of Matrigel and gelatin substrata for feeder-free culture of undifferentiated mouse embryonic stem cells for toxicity testing.

Murine embryonic stem (mES) cells have been used to evaluate cytotoxicity and developmental injury following exposure to embryotoxic agents. However, maintaining a homogeneous population of undifferentiated mES cells for this purpose has been complicated by the need for continuous co-culture with murine embryonic fibroblast (mEF) cells or limited passaging on plastic surfaces coated with gelatin. Here, we compare the synthetic basement membrane Matrigel with 0.1% gelatin substratum for feeder-free propagation of undifferentiated mES cells. Biomarkers of pluripotentiality, chromosome number, caspase-3 expression, and cardiomyocyte differentiation were monitored for mES cells cultured on Matrigel or 0.1% gelatin up to passage 7 (P7). Our results suggest that choice of substratum had no significant effect on population doubling time, cell viability, stage-specific embryonic antigen-1 (SSEA-1) expression, or early passage formation of beating cardiomyocytes (all P>or=0.09). In other comparisons, however, Matrigel supported significantly higher synthesis of alkaline phosphatase (7.7x10(-3)+/-0.8 vs 6.6x10(-3)+/-0.8 units/liter/cell, respectively, P=0.012), overall expression of activated caspase-3 following exposure to 5, 10, 50, 100 and 500 parts per billion (ppb) sodium arsenite (P<0.0001), and percent development to beating cardiomyocytes at P7 (P=0.01). Together, our findings suggest that Matrigel shows promise as a substrate for feeder-free propagation of undifferentiated mES cells for embryotoxicity endpoints.

Combined effects of Matrigel and growth factors on maintaining undifferentiated murine embryonic stem cells for embryotoxicity testing.

Undifferentiated, murine embryonic stem (mES) cells have shown promise as a substrate for identifying embryotoxic chemicals and for studying mechanisms of early developmental injury. However, long-term maintenance of mES cells in an undifferentiated state is problematic. The present study evaluates the combination of Matrigel matrix and three growth factors for this purpose. Biomarkers of mES cell pluripotency, apoptosis, chromosome number and cardiomyocyte differentiation were monitored over 119 population doublings. D3 mES cells retained undifferentiated characteristics, including sustained expression of alkaline phosphatase and stage specific embryonic antigen-1 (SSEA-1) and continued transcription of Pou5f1 (Oct-4). Cell viability remained at > or=95% and population-doubling times averaged 14.3 h over 10 weeks of observation. Caspase-3 activation, a marker of cellular death by apoptosis, was measured in early- and late-passage mES cells. Early-passage cells showed dose-responsive caspase-3 activation following exposure to sodium arsenite, whereas caspase-3 activation of late-passage cells dropped to background levels at toxicant dosages above 50 ppb. Aneuploidy and impaired differentiation into beating cardiomyocytes were noted for late-passage mES cells. Matrigel, combined with growth factors, may sustain undifferentiated mES cells. However, aneuploidy, reduced caspase-3 activation, and inability to differentiate suggests further modifications to the culture system may be needed for long-term propagation of cells for embryotoxicity endpoints.

A study of the temporal relationship between plasma high molecular weight angiotensinogen and the development of pregnancy-induced hypertension.

In addition to the normally prevalent low molecular weight angiotensinogen (LMrA), significant quantities of a high molecular weight angiotensinogen (HMrA) are present in the human pregnant state. Previous studies have documented that 47% of women who develop pregnancy-induced hypertension (PIH) have a significantly elevated plasma HMrA/LMrA ratio. The purpose of this study is to establish whether or not the increase in the HMrA/LMrA ratio precedes the development of hypertension. Serial plasma samples were collected from a group of women throughout their pregnancy. High molecular weight angiotensinogen and LMrA levels in the samples from these women were determined. Fifteen of these women developed PIH. Seven women in the PIH group had a significantly elevated plasma HMrA/LMrA ratio. There was no consistent relationship between the elevation of the HMrA/LMrA ratio and the onset of hypertension. Three women had an elevated HMrA/LMrA ratio before the development of hypertension. In one woman the two events occurred simultaneously, and in three women the HMrA/LMrA ratio was elevated only after the development of hypertension. The current study shows that the development of hypertension during pregnancy is not the primary biologic signal for elevation of the plasma HMrA/LMrA ratio. Other parameters associated with fetal distress or abnormal development of placental circulatory systems must be involved in increasing the HMrA/LMrA ratio. It is proposed that the elevation of the HMrA/LMrA ratio is a mechanism by which the placental tissue specific renin-angiotensin system is attenuated.

Quantitation of the five forms of plasma high molecular weight angiotensinogen in women with pregnancy-induced hypertension.

In the human pregnant state a high molecular weight form of angiotensinogen (HMrA) is present in significant quantities in addition to the usual low molecular weight angiotensinogen (LMrA). In a previous study involving a small number of white women, it was found that women who had developed pregnancy-induced hypertension (PIH) had significantly higher levels of plasma HMrA. It has been determined that there are five isoforms of HMrA. The objectives of this study were to expand the previous study with the inclusion of black women and to determine which isoform(s) of plasma HMrA are elevated in PIH. Plasma LMrA and HMrA were quantitated in 24 normotensive pregnant women and 65 women with PIH. The PIH group had higher levels of HMrA and somewhat lower levels of LMrA than the normotensive group. The HMrA/LMrA ratio was elevated in 47% of the PIH group. The five isoforms of HMrA were quantitated in plasma from 10 white women with PIH, 10 black women with PIH, and 6 normotensive pregnant white women. Half of both the white and black women with PIH had an elevated HMrA/LMrA ratio. The relative proportion of the HMrA isomers was similar in all groups. These studies show that half the women with PIH have a distinct abnormality in their renin angiotensin system. Both white and black women show this abnormality. In those women who have an elevated total HMrA, all five isoforms of HMrA are equally elevated.