Aims:  This research investigated the effect of sonication at frequencies of 20, 40 and 580 kHz and approximately the same acoustic intensity on the viability and declumping of two micro‐organisms (Escherichia coli and Klebsiella pneumonia). Methods and Results:  Two analytical methods were employed; viable plate counts (CFU ml−1) and flow cytometry to identify and quantify both live/viable and dead bacteria in the bulk liquid. Flow cytometry results for E. coli and Kl. pneumonia indicated a high sensitivity to 20 and 40 kHz frequency with a continuous decrease in the viable cells and an increase in dead cells during experiments. In contrast, results using the higher frequency of 580 kHz indicate predominantly deagglomeration of bacterial clumps rather than cell membrane disruption (Joyce et al. 2003). Results indicate a good correlation between flow cytometry and viable plate count methodology. Conclusions:  Sonication has two different effects on bacteria (i) inactivation and (ii) declumping; however, the scale of these effects is dependent on intensity and frequency. Flow cytometry provides a method to distinguish between and quantify the effects through the observation of two subpopulations: (i) live/viable and (ii) dead bacterial cells. Significance and Impact of the study:  Treatment using power ultrasound has been shown to have a significant impact on microbial activity. This is the first time a study has compared the influence of a range of different frequencies, but at similar power settings on the survival of bacteria in phosphate buffer saline (PBS). This work is of importance for applications where ultrasound has been considered for use in industry as a means of disinfection including the treatment and pretreatment of water and also for the sterilization of liquid foods.

The aim of this study is to assess the synergetic effect of combined ultrasound and ozone treatment on the biological disinfection of water on a large-scale application using viable plate counts and flow cytometry. Escherichia coli B bacteria in saline suspension was treated using a commercially available combined ultrasound and ozone system (USO3 (Ultrasonic Systems Gmbh)) for 16 min. Two analytical methods were used to assess the results in terms of live and dead cells in the bulk liquid: standard viable plate counting recorded in terms of colony forming units per milliliter and flow cytometry. In the latter case 1 mL of bacterial suspension was stained simultaneously with the fluorescent stains SYTO9 and propidium iodide (PI). Transmission electron microscopy was used to generate images identifying the biological effects of different treatments using ultrasound and ozone on bacterial cell walls. Results demonstrated that treatment with ozone alone (1 mg/L) resulted in a significant reduction (93%) in the number of live cells after 16 min treatment whereas ultrasound alone showed only a small reduction (24%). However, a combination of ozone and ultrasound showed a synergistic effect and enhanced the inactivation to 99% after 4 min. A combined ultrasound and ozone treatment of bacterial suspensions using a commercial system affords a promising method for water disinfection that is better than treatment using either method alone. Standard viable plate count analysis is normally used to assess the effectiveness of disinfection treatments; however flow cytometry proved to be a more sensitive method to determine the actual effects in terms of not only live and dead cells but also damaged cells. This type of analysis (cell damage) is difficult if not impossible to achieve using traditional plate counting methodology.

Acinetobacter baumannii received attention for its multi-drug resistant associated with many severe infections and outbreaks in clinical environment. The aims of the study are to investigate the antibiotic susceptibility profile of clinically isolated A. baumannii, biofilm production, and the efficiency of Low Frequency Ultrasound (LFU) and honey to attenuate biofilm production. A total of 100 samples were taken from different sources from Baghdad hospitals. The susceptibility patterns revealed the percentage of pan drug resistant (PDR) isolates were 1.5 %, 72.7 % were extended drug resistant (XDR), 16.7 % were multidrug resistant (MDR), and 9.1 % were non MDR and sensitive to most antibiotics used. The ability to form biofilm was detected by crystal violet staining, and the results showed that 20% were strong biofilm, and 31.8% were moderate biofilm. The biofilm formation percentage was decreased using Low frequency ultrasound LFU and honey. Moreover, PCR results revealed that not all of them harbouring biofilm-related genes or integrons (bap, csuE, IntI-1, IntI-2), although, they are strong biofilm producers. These results conclude that low frequency ultrasound and chemical components of honey might be a good choice to restrain A. baumannii biofilm formation, and negative correlation between antibiotic resistance and biofilm ability. Running title: Antimicrobial drug resistance, Acinetobacter baumannii, biofilm inhibition