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Acinetobacter PDF Print E-mail
Sunday, 04 April 2010 00:00
Lynne Leach
Luis Actis

  • Acinetobacter spp. are ubiquitous in the environment
  • facilitate coagulation in waste-water treatment
  • form biofilms
  • multi-drug resistant strains are becoming an important source of nosocomial infections
Acinetobacter are aerobic, non-motile, gram negative bacteria that are ubiquitous in the environment and have been identified in drinking water, sewage water, groundwater, dental lines, rivers, soil,  human skin, vegetables and fruits, ponds and swamps ( Baumann P 1968; Warskow and Juni 1972; Bifulco et al. 1989; Tall et al. 1995; Barbeau et al. 1996; Ford TE 1999; Fournier and Richet 2006). Although Acinetobacter are not generally considered pathogenic, the A. baumannii - A. calcoaceticus complex is increasingly associated with nosocomial infections in compromised patients. There have been 128 Acinetobacter clinical outbreaks between 1977 and 2006 ( Fournier and Richet 2006; Seifert and Wisplinghoff 2008). Among the clinically relevant strains, A. baumannii, gen. sp3, and gen.sp13TU, are the most predominant isolates ( Wroblewska et al. 2004; Dijkshoorn et al. 2007). Acinetobacter have been associated with respiratory infections, wound infections, bacteremia, secondary meningitis, and urinary infections ( Bergogne-Berezin and Towner 1996; Wroblewska et al. 2004; Keum et al. 2006; Seifert and Wisplinghoff 2008). In immuno-compromised patients mortality rates can be as high as 64%  ( Seifert and Wisplinghoff 2008). One of the reasons for this high mortality rate is the difficulty clearing Acinetobacter infections, as 10-33% of Acinetobacter strains isolated in hospital settings are multi-drug resistant ( Takahashi et al. 2000; Ecker et al. 2006; Coelho et al. 2006; Rodriguez-Baño et al. 2006; Fournier and Richet 2006; Turton et al. 2006a; Turton et al. 2006b; Dijkshoorn et al. 2007; Towner KJ 2008; Seifert and Wisplinghoff 2008). Multi-drug resistant A. baumannii has become a critical pathogen in wounded U.S. soldiers returning from Iraq and Afghanistan ( Dijkshoorn et al. 2007). Another reason for the difficult elimination of Acinetobacter spp. might be their ability of biofilm formation, which might also contribute to greater resistance to disinfection in the hospital setting ( Vidal et al. 1996; Tomaras et al. 2003; Shakeri et al. 2007; Loehfelm et al. 2008).  Water supplies might be an important mode of hospital contamination as Acinetobacter is often part of the profile of waterborne heterotrophic bacteria ( Stewart and Rochelle 2006). While Acinetobacter do not typically pose a concern for the general public, these bacteria are emerging pathogens in the hospital setting, and therefore warrant concern in drinking water treatment standards. 
Acinetobacter spp. are ubiquitous in environmental and treated waters, and have been found in sewage water, groundwater, surface water, and drinking water ( Baumann P 1968; Warskow and Juni 1972; Bifulco et al. 1989; Stewart and Rochelle 2006). Acinetobacter strains have been isolated in 97% of natural surface waters in numbers up to 100 cells/ml ( WHO 2006). In distributed water 5-92% of samples tested positive, and may constitute between 1-5.5% of heterotrophic plate count (HPC) flora in drinking water samples ( WHO 2006).

Examples of detection:

  • Acinetobacter were found in 38% (n=63) of groundwater samples by cultivation. The samples had a mean density of 8 cfu/100 ml ( Bifulco et al. 1989).
  • By bacterial culture methods, it has been estimated that Acinetobacter composes at least 0.001% of the total aerobic heterotrophic population in water and soil ( Baumann P 1968).
  • A. calcoaceticus composed 23% of the culturable species from a dental unit water line. The contamination was suspected to originate from the public drinking water distribution system as opposed to contamination from an oral source ( Barbeau et al. 1996).
  • Eleven days after the installation of a dental water supply line leading to an air-water syringe A. calcoaceticus was isolated from the newly formed biofilm ( Tall et al. 1995).
  • Using culture based methods, Acinetobacter strains were isolated from: soil (26/105), surface water (56/105), and sewage (23/105) ( Warskow and Juni 1972).
  • The isolation frequencies from various environmental sources of Acinetobacter isolated on the basis of their ability to degrade hydrophobic substances were: 31/58 from soil, 10/58 from fresh and seawater, and 11/58 from sludge or activated sludge ( Gutnick and Bach 2008).
Acinetobacter spp. have been reported to have similar disinfection susceptibilities to other heterotrophic bacteria ( WHO 2006; Stewart and Rochelle 2006). Increased resistance to chlorine, chloramine and chlorine dioxide have been indicated in Acinetobacter cell aggregates ( Stewart and Rochelle 2006). There is evidence that A. calcoaceticus causes aggregation, and therefore may promote the formation of mixed-species biofilms ( Malik et al. 2003; Simões et al. 2008). In general, biofilms in the distribution system are much more resistant to disinfection than planktonic cells, and Acinetobacter have been found in drinking water distribution system biofilms ( Ford TE 1999; Simões et al. 2007b). An A. calcoaceticus strain isolated from drinking water was shown to form biofilms on stainless steel, copper, polypropylene, polyethylene and silicone ( Simões et al. 2007a). The promotion of biofilm formation by Acinetobacter may also be significant in harboring frank pathogens in the drinking water distribution system.

Selected studies on susceptibility to disinfection are summarized in the following:

  • A four-species biofilm including: Microbacterium phyllosphaerae, Shewanella japonica, Dokdonia donghaensis, and A. lwoffii was found more resistant to hydrogen peroxide and tetracycline than any of the single-species biofilms ( Burmølle et al. 2006). This may have implications in the drinking water distribution system.
  • A. johnsonii A2 was isolated from a high altitude Andean Lake exposed to strong UV irradiation. Survival rate of the isolate upon exposure to artificial UV-B irradiation (3,931 J m-2) was 48%. The survival rate of the control strain, A. johnsonii ATCC 17909, was 20%. However, culturability of both strains was fully recovered to pre-treatment levels after exposure to photosynthetic active radiation (400-700 nm) and UV-A (315-340 nm), which induces the photo-reactivation DNA repair mechanism ( Zenoff et al. 2006). 
  • The resistance to different chemicals was determined for a strain of A. lowffi that was isolated from a pharmaceutical water purification system. The D value, defined as the time in minutes necessary to kill 90% of cells, was determined in (1) 0.5% citric acid, D = 1.77; (2) 0.5% hydrochloric acid, D = 7.26; (3) 70% ethanol, D = 6.84; (4) 0.5% sodium bisulfite, D = 4.82; (5) 0.4% sodium hydroxide, D = 12.21; (6) 0.5% sodium hypochlorite, D = 5.14; (7) 2.2% hydrogen peroxide and 0.45% peracetic acid, D = 4.19 ( Mazzola et al. 2006). The authors also reported data for Pseudomonas aeruginosa, Pseudomonas fluorescens, Pseudomonas alcaligenes, Pseudomonas picketti, Flavobacterium aureum, and Pseudomonas diminuta, however, this is beyond the scope of this review to report.
  • In a membrane filtration assay, 3.3% of Acinetobacter strains isolated from DWDS survived exposure to 1.0 mg/L free chlorine. None of the cells survived 10 mg/L free chlorine. This study also indicated that Acinetobacter cells isolated from a DWDS with residual chlorine were more resistant to combined chlorine ( Ridgway and Olson 1982).
  • Acinetobacter strains isolated from a cooling water system were found to be strong biofilm formers, both in single-species and mixed-species biofilms. To test the disinfectant efficacy, the biofilms were allowed to form for 24 hours and were then exposed to disinfectant.  Hypochlorite and hydrogen peroxide together were more effective in removal and killing than exposure to the combination of sulfathiazole and glutaraldehyde. The mixed-species biofilm tended to be more resistant than the single-species Acinetobacter biofilm ( Shakeri et al. 2007). 
  • Planktonic cells of Acinetobacter 1-19 were able to withstand 250 mg/L copper, but the formation of a single-species biofilm was inhibited ( Mogilnaya et al. 2008).
Despite their ubiquitous presence in water, little information is available on the survival of Acinetobacter in water. Generally, Acinetobacter is resistant to desiccation, can grow on a broad spectrum of substrates, and form biofilms ( Dijkshoorn et al. 2007). Acinetobacter spp. were reported to survive for up to 6 days on dry filter paper ( Bergogne-Berezin and Towner 1996). Furthermore, A. baumannii was shown to survive for approximately 27 days on a dry glass cover slip ( Jawad et al. 1998a). Acinetobacter have also been shown capable of long term survival on hospital equipment including tap water faucets, ventilators, and bedside urinals, beds, and pillows ( Villegas and Hartstein 2003). Survival in the drinking water distribution system might be enhanced by the ability of Acinetobacter to form biofilms (section on susceptibility to disinfection).

  • Using a membrane sandwich method, Acinetobacter cells (200 cells/ml) were submerged in either oligotrophic lake water or distilled water kept at 10ºC. Less than 10% of Acinetobacter cells could be recovered by culture after 24 hours in either the distilled or lake water ( Sjogren and Gibson 1981).
  • Acinetobacter were isolated from macroscopic microbial streamers found in the Rio Tinto, an extremely acidic river environment in Spain ( García-Moyano et al. 2007). 
The infectious dose of Acinetobacter spp. in humans is unknown, but these bacteria are generally considered low virulence. However, Acinetobacter are increasingly being associated with hospital-acquired infections. A. baumannii has a 50% lethal dose of 106-108 cells when inoculated intraperitoneally in neutropenic mice ( Bergogne-Berezin and Towner 1996). Results from Eveillard et al. 2010 show that virulence can vary greatly between strains of A. baumannii.  Some factors that are associated with virulent strains of Acinetobacter include the presence of a capsule, adhesion to human epithelial cells, production of lipid degrading enzymes, the presence of lipid A and the production of the outer membrane protein OmpA ( Bergogne-Berezin and Towner 1996; Stewart and Rochelle 2006; Gaddy et al. 2009).
Molecular methods specifically for the detection of Acinetobacter in drinking water systems are non-existent. The majority of molecular methods available are used to distinguish and track epidemic outbreaks of the Acinetobacter calcoaceticus - Acinetobacer baumanni complex in clinical settings. These techniques include: pulsed-field gel electrophoresis (PFGE) ( Seifert et al. 2005), Ribotyping ( Gerner-Smidt P 1992; Seifert and Gerner-Smidt 1995), AFLP( Dijkshoorn et al. 1996), amplified ribosomal DNA restriction analysis (ARDRA) and restriction fragment length polymorphism (RFLP) ( Jawad et al. 1998b), tRNA spacer fingerprinting ( Wiedmann-al-Ahmad et al. 1994; Ehrenstein et al. 1996), 16S-23S spacer fingerprinting ( Chang et al. 2005), repetitive extragenic palindromic sequence-based PCR (REP-PCR) ( Snelling et al. 1996). An excellent summary of these techniques is provided in the recent book Acinetobacter Molecular Biology ( Dijkshoorn and Nemec 2008; Seifert and Wisplinghoff 2008).

Some of the following molecular methods developed may be adaptable for application to drinking water monitoring:

  • Real-time PCR: A real-time PCR method has been developed to detect Acinetobacter in the bloodstream ( Wellinghausen et al. 2004).  In this assay 16S eubacterial primers were used with a specific Acinetobacter TaqMan probe. This PCR protocol was designed specifically on the sequence of A. lwoffii ATCC 15309 and A. junii ATCC 17908.  The detection limit was 10 pg of genomic DNA per PCR reaction for either strain; the diagnostic sensitivity was 100% (10/10). This may well be a method that can be transferred to screening drinking water samples. There exists a reference for an unpublished method for a TaqMan assay for Acinetobacter ( Rajal et al. 2007).
  • PCR: The integrase gene has been used as a PCR-target for discrimination between strains. This was effective for identifying potentially pathogenic strains of the bacteria, but has not been tested in drinking water ( Turton et al. 2005).
  • Multiplex PCR: Multiplex PCR targeting two antibiotic resistance genes and an integrase gene has been used for the detection of A. baumannii in clinical specimens ( Turton et al. 2006b). This may be a reasonable technique for screening drinking water, as A. baumannii appears to be the predominant pathogenic strain of Acinetobacter.
  • PCR/ESI-MS: Multilocus PCR combined with electrospray ionization mass spectrometry (PCR/ESI-MS) was used to simultaneously identify and genotype strains of Acinetobacter. In this method, PCR amplification of several loci were distinguished with the use of ESI-MS. ESI-MS can differentiate based on nucleotide composition, as apposed to DNA sequencing. This technique has the advantage of being very rapid, however, it requires the use of advanced equipment which is not always readily available. As it stands, the development of this technique has focused on genotyping of A. baumannii ( Ecker et al. 2006). Further optimization of this technique may allow for environmental identification of Acinetobacter.
  • FISH: Fluorescence in situ hybridization (FISH) has been used to track Acinetobacter in sewage sludge. The probe was designed to target the 16S rRNA gene ( Wagner et al. 1994; Liu et al. 2005).
  • Microarray: A microarray technique, targeting the 23S rRNA gene, was developed for the detection of A. baumanii in clinical samples. The technique has a sensitivity of 84.6% (11/13) and a specificity of 100% ( Keum et al. 2006).
The following pictures are part of the Public Health Image Library (PHIL) from the Centers for Disease Control and Prevention (CDC). The photos are in the public domain and thus free of copyright restrictions. We would like to express our appreciation for providing these images.


Figure 1:

                                               Acinetobacter_photo 1

This SEM depicts a number of clusters of aerobic Gram-negative, non-motile Acinetobacter baumannii bacteria under a relatively low magnification of 1,546x.

Source: http://phil.cdc.gov/phil/details.asp

Photo ID: 10096

Content provider(s): Centers for Disease Control and Prevention/ Janice Haney Carr
Credit: Janice Haney Carr


Figure 2:

                                                Acinetobacter_photo 2

This SEM depicts a highly magnified cluster of Gram-negative, non-motile Acinetobacter baumannii bacteria; Mag - 27600x.

Source: http://phil.cdc.gov/phil/details.asp

Photo ID: 10095

Content provider(s): Centers for Disease Control and Prevention/ Matthew J. Arduino
Credit: Janice Haney Carr


Figure 3:

                                                 Acinetobacter_photo 3

This SEM depicts a highly magnified triad of Gram-negative, non-motile Acinetobacter baumannii bacteria; Mag - 24730x.

Source: http://phil.cdc.gov/phil/details.asp

Photo ID: 6499

Content provider(s): Centers for Disease Control and Prevention/ Matthew J. Arduino, DRPH; Janice Carr; Jana Swenson
Credit: Janice Carr

Last Updated on Tuesday, 20 September 2011 13:30


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