Descriptive Cross-Sectional Study on Acanthamoeba - Associated Pseudomonas Species at Kenyatta National Hospital Intensive Care Unit

Free-living amoeba (FLA) such as Acanthamoeba spp. are ubiquitous protozoa that graze on microorganisms such as bacteria, viruses and algae. However, amoebic resistant microorganisms (ARM) such as Pseudomonas spp. evade being killed by amoeba thus multiplying within the free-living amoeba. Free-living amoeba may therefore enhance transmission of Pseudomonas spp. across hospital environments thus contributing to the burden of nosocomial infections and spread of antimicrobial resistance. In Kenya, nosocomial Pseudomonas spp. infections present a major health challenge but the role of free-living amoeba such as Acanthamoeba spp. in transmission of these infections has so far not been assessed. This study aimed at the isolation of Acanthamoeba spp. from various surfaces and equipment and molecular detection of the Acanthamoeba-associated Pseudomonas spp. spp. on various surfaces and equipment at KNH ICU. This suggests a possible role of Acanthamoeba spp. in Acanthamoeba- ARM associated nosocomial infections transmission and in spread of antimicrobial resistant genes across microorganisms in hospitals.

Acanthamoeba spp. existence in hospital environment pose an explicit risk of expedient infections to the immunocompromised patients such as granulomatous amoebic encephalitis (GAE), sinusitis and skin lesions (Barnard, 2015;Shokri et al., 2016;Taravaud, Loiseau and Pomel, 2017;Souza, 2018). Amoebic keratitis is manifested among the immunocompetent individuals commonly in the contact lens users, patients with corneal injury and those living in places with inadequate water supply (Chappell et al., 2001;Khan, 2003;Booton et al., 2010;Clarke et al., 2012;Muchesa et al., 2014). Besides, Acanthamoeba spp. have been associated with more than 100 species of pathogenic bacteria which may be transmitted to humans and higher animals (Douesnard-Malo and Daigle, 2011; Gryseels et al., 2012;Aç et al., 2013;Fabres et al., 2016). Pseudomonas sp., a known ARM with high antibiotic and disinfectants resistance, increased virulence is also an etiologic agent of nosocomial infections (Lim and Webb, 2005).
The ubiquity of Pseudomonas sp. allows for its vast spread to patients from diverse sources including air, food, water, visitors, linen, contaminated medical personnel, contaminated surfaces and equipment such as catheters and ventilators which readily predisposes patients to nosocomial infections (Davane et al., 2014). Pseudomonas has been isolated from bacterial cultures as an etiologic agent of primary infections and/ or nosocomial infections from patients at Kenyatta National Hospital Intensive Care Unit (KNH ICU) (Njoki, 2009).
Nonetheless, since most infections are linked to well-known free pathogens, the role of FLA such as Acanthamoeba spp. and ARMs such as Pseudomonas spp. in disease burden is mostly overlooked . This could be one of the reasons for the escalated rates of nosocomial infections despite stringent infection control measures (Altayyar et al., 2016). This is further complicated by the lack of information on FLA, ARMs and the possible effects of their interactions and the lack of awareness among healthcare personnel in Kenya. The study therefore sought to assess the prevalence of Acanthamoeba sp. and associated Pseudomonas sp. from selected surfaces and equipment in ICU at Kenyatta National Hospital (KNH), the largest teaching and referral hospital in East and Central Africa located in Nairobi, the capital city of Kenya, in order to inform infection control policy.

Methods
This was a cross-sectional study in which Acanthamoeba spp. and free bacteria were cultured and isolated from swabs collected from selected surfaces and equipment in ICU at KNH, and detection of Pseudomonas sp. genomic DNA within the isolated Acanthamoeba spp. using PCR was done. Surfaces and equipment in the hospital ICU that could potentially act as fomites were identi ed and swabs collected.
Specimen collection, culture and isolation Swabs were aseptically collected from selected surfaces and equipment in duplicate and immediately delivered to the University of Nairobi Medical Microbiology Laboratory. Bacterial cultures were performed on one batch of swabs on Blood Agar (BA) and MacConkey (MAC) agar using the streak plate procedure and aerobically incubated at 37 0 C for 18 to 24 hours (Sanders, 2012). Plain plates of BA and MAC were used as negative controls and Pseudomonas sp. ATCC 27853 inoculated on BA and MAC plates were used as positive controls under the same conditions. Bacteria identi cation was based on colonial morphology, haemolysis on BA, lactose fermentation, Gram stain results and biochemical tests (Bisen et al. 2012).
Acanthamoeba spp. culture and detection of ARMs from the second batch of swabs was done as described by others (Lagier et al. 2015). Acanthamoeba castellani trophozoites (ATCC 30010) were cultured on Non-Nutrient Agar (NNA) media as positive control and to con rm the suitability of NNA for its intended use (Kara et al., 2015). The swabs for use in Acanthamoeba sp. culture were suspended in 2 milliliters (ml) sterile Page saline in 13x100mm tubes and centrifuged at 1000 revolution per minute (RPM) for 10 minutes ( PCR detection of Pseudomonas sp. genomic DNA Detection of Pseudomonas sp. genomic DNA was done using PCR (Spilker et al., 2004;Paulo, 2015). The primers PA-GS-F (5'-GACGGGTGAGTAATGCCTA-3') and PA-GS-R (5'-CACTGGTGTTCCTTCCTATA-3') were used to amplify 618 base pairs fragment of the Pseudomonas sp. genomic DNA. Agarose gel electrophoresis of the ampli ed products was run as described by others (Sambrook and Russell, 2006;Kafkas et al., 2012). Pseudomonas sp. ATCC 27853 as positive control bands were used to locate and identify ampli ed Pseudomonas sp. genomic DNA extracted from Acanthamoeba spp. isolates.
A total of 153 swabs were cultured on NNA for the isolation of Acanthamoeba spp. Almost all 143 (93.5%) of swabs were positive for Acanthamoeba spp. Acanthamoeba spp. (Figs. 1 and 2) growth was positively associated with the swab location; P = 0.008 (χ 2 = 71.160 df 45). Subsequent sub-cultures to purify Acanthamoeba spp. isolates was done on 96 of the primary Acanthamoeba spp. cultures of which only 22 (22.9%) were successful. There was a considerable drop in the proportion of positive Acanthamoeba spp. subcultures (Table 1).

Discussion
The study, aiming at isolating Acanthamoeba spp. and detecting Pseudomonas sp. genomic DNA within the isolated Acanthamoeba spp. from selected surfaces and equipment was conducted in the ICU at Kenyatta National Hospital. To the best of our knowledge this was the rst study on Acanthamoeba spp. and the associated ARMs in Kenya.  Invasion of and survival within FLA by bacteria is enhanced by effector proteins secreted by Type III or Type IV secretory systems expressed by majority of amoeba resistant bacteria (ARB). The released effector proteins manipulate FLA defense system in the favour of the ARB. This has been reported in Pseudomonas aeruginosa which expresses Type III secretory system that releases effector proteins capable of lysing FLA (Kara et al., 2015). This could partly explain the drop in the number of positive Acanthamoeba spp. subcultures from primary cultures assuming that effector proteins released by Pseudomonas spp. could have lysed majority of primary Acanthamoeba spp isolates.
The detection of Pseudomonas sp. genomic DNA within Acanthamoeba spp. isolates con rms a potential health risk to patients at KNH ICU should these patients acquire ARM infections. This is because the mechanisms used by ARMs to evade amoebic killing could also be used to evade macrophage killing (Balczun and Scheid, 2017). Also, in the event that drug resistant bacteria coexist with drug susceptible bacteria within the same Acanthamoeba sp. host, the Acanthamoeba sp. could become a hot spot of drug resistant gene transfer which could end up increasing the spread of drug resistant bacteria (Bertelli and Greub, 2012;Fukumoto et al., 2016). The study con rmed the co-existence of Pseudomonas spp. within Acanthamoeba spp in a hospital set up. A study in South Africa that detected ARMs within FLA suggested the latter could act as survival and proliferation niches from where ARMs can be transmitted to immunosuppressed patients in hospitals (Muchesa et al., 2017). Although there was no statistically signi cant association between swab location and detection of Pseudomonas sp. genomic DNA, the present study con rms that Acanthamoeba spp. are ubiquitous and raises a concern for intensive care unit patients.  (Costa et al., 2010). This is important considering that the disinfectants currently used to clean surfaces in many health facilities are not effective against FLA such as Acanthamoeba spp. (Iqbal, Siddiqui and Khan, 2014). Consequently, control measures against the spread of drug resistant pathogenic bacteria could also be implicated especially in hospital setting.

Conclusion
The ndings of this study demonstrate that Acanthamoeba sp., a free-living amoeba proliferates on various important surfaces and equipment in the ICU at KNH despite routine cleaning activities. This is most likely to be happening not only in other units of the hospital but in other hospitals as well. The study also demonstrates that Acanthamoeba spp. in the ICU may harbor and act as protected niche for potentially pathogenic bacteria such as Pseudomonas sp. from where the bacteria are re-introduced in the environment thus frustrating nosocomial infection control efforts. This calls for design and implementation of more rigorous cleaning, disinfection and sterilization strategies that are effective against FLA. Drug resistance in pathogenic bacteria is a major global health challenge and future studies to shed light on the role of Acanthamoeba spp. and other FLA in the spread of the antimicrobial drug resistance are needed.