Occurrence of environmental Acinetobacter baumannii similar to clinical isolate in

AEM Accepts, published online ahead of print on 28 February 2014 Appl. Environ. Microbiol. doi:10.1128/aem.00312-14 Copyright 2014, American Society for Microbiology. All Rights Reserved. 1 2 Occurrence of environmental Acinetobacter baumannii similar to clinical isolate in paleosol from Croatia 3 4 Jasna Hrenovic, a # Goran Durn, b Ivana Goic-Barisic, c Ana Kovacic d 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 University of Zagreb, Faculty of Science, Division of Biology, Zagreb, Croatia a ; University of Zagreb, Faculty of Mining, Geology and Petroleum Engineering, Croatia b ; Department of Clinical Microbiology, University Hospital Centre Split and University of Split School of Medicine, Split, Croatia c ; Institute of Public Health of Split and Dalmatia County, Split, Croatia d Running Head: Occurrence of environmental Acinetobacter baumannii #Address correspondence to Jasna Hrenovic, jasna.hrenovic@biol.pmf.hr Over the last decade bacteria of the genus Acinetobacter have emerged as a leading cause of hospital acquired infections. Outbreaks of Acinetobacter infections are considered to be caused exclusively by contamination and transmission in hospital environment. Natural habitats of clinically important multiresistant Acinetobacter sp. remain to be defined. In this paper we report an incidental finding of viable clinically related multidrug resistant strain of Acinetobacter baumannii in acid paleosol from Croatia. The environmental isolate of A. baumannii showed 87% of similarity with a clinical isolate originating from hospital in this geographic area and was resistant to gentamicin, trimethoprim/sulfamethoxazole, ciprofloxacin and levofloxacin. In paleosol the isolate was able to survive low ph (3.37), desiccation and high temperature (50ºC). The 1

26 27 28 probable source of A. baumannii in paleosol is an illegally disposed waste of different origin situated in the abandoned quarry. The bacteria could have been leached from waste by storm water and infiltrated in paleosol. 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 Bacteria of the genus Acinetobacter have been recognized as significant hospital pathogens since the late 1970s, but at the time they were easily treated as they were susceptible to commonly used antimicrobials. Acinetobacter sp. has an increasing ability to develop antimicrobial resistance to commonly used antimicrobial agents, leading to limited options for antibiotic treatment (1). Three major overlapping populations of bacteria of the genus Acinetobacter are known: hospitals and hospitalised patients multiresistant isolates (A. baumannii, Acinetobacter genomic species 3 recently renamed as Acinetobacter pittii, and genomic species 13TU recently renamed as Acinetobacter nosocomialis; all three known as A. baumannii complex); skin (humans and animals) and foodstuff sensitive isolates (A. johnsonii, A. lwoffii, A. radioresistens); soil and wastewater sensitive isolates (A. calcoaceticus, A. johnsonii) (2). Over the last decade bacteria of the genus Acinetobacter have emerged as a leading cause of hospital acquired infections, where A. baumannii accounts for approximately 80% of all reported Acinetobacter infections (3). A. baumannii is among the pathogens targeted in the call to develop new antibiotics by 2020 (4). Problems caused by multi-drug resistant Acinetobacter baumannii in hospital settings are emphasised by their increasing ability to develop antimicrobial resistance to commonly used antimicrobial agents, ability to form biofilm on abiotic surfaces, and high degree of resistance to drying and disinfectants, leading to long-term persistence in the hospital environment (2). Possible sources for occurrence of outbreaks include: preinjury skin colonization, introduction from the environment at the time of injury, and acquisition after injury during treatment in hospitals (5). Contamination of hospital environment accompanied 2

51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 66 67 68 69 70 71 72 73 74 75 with the transmission of infection within the hospital is generally recognised as the major source in occurrence of outbreaks (6). According to the Bergey s manual of systematic bacteriology (7) bacteria of the genus Acinetobacter are saprophytes occurring naturally in soil, water, sewage and food. This statement has been cited in the introduction part of papers dealing with the clinical isolates of Acinetobacter sp. Such previous literature reports of ubiquitous presence of clinically important Acinetobacter sp. in natural environments, such as soil and water, are nowadays recognized as misconceptions (1). The prevalence of clinically important Acinetobacter sp. in the nature and their potential to migrate in and/or out of the hospital environments is undefined up to date. Natural habitats of clinically important multiresistant Acinetobacter sp. remain to be defined. Colonization of digestive tract of patients with multi-drug resistant Acinetobacter sp. in hospitals occurs in high rates (8). Digestive tract of colonized patients could be an important epidemiologic reservoir of multiresistant Acinetobacter sp., from which bacteria could migrate through wastewater in the environment. The Istrian peninsula represents the NW part of spacious Adriatic Carbonate Platform (9). This part of the platform is composed of a succession of carbonate deposits more than 2000 m thick, of Middle Jurassic (Bathonian) to Eocene age, and is overlain by Palaeogene (Eocene) Foraminiferal limestones, Transitional beds (Globigerina marls) and flysch deposits. Carbonate deposits of the Istrian Peninsula exhibit numerous exposure surfaces reflecting emergence. Greenish-grey clays associated with Late Aptian-Late Albian regional emergence show clear evidence of subaerial exposure and are considered paleosols (10, 11). In the Tri jezerca quarry (Fig. 1) they are situated in palaeokarst pits of the Lower Aptian massive limestones, which is exploited as a building-stone known under the name of Istarski žuti (Istrian Yellow). This stone is presently exploited in a few quarries in central Istria. However, 3

76 77 78 79 abandoned part of Tri jezerca quarry is currently used as an illegal landfill. Disposed waste of different origin found in this abandoned quarry may be an important threat to the environment. In this paper we report an incidental finding of viable clinically related multidrug resistant strain of A. baumannii in paleosol from Istria, Croatia. 80 81 82 83 84 85 86 87 88 89 90 91 92 93 94 95 96 97 98 99 100 MATERIALS AND METHODS Characterization of paleosol. Greenish-grey clays in the Tri jezerca quarry are up to 93 cm thick (Fig. 2). The paleosol profile was carefully cleaned and prepared for sampling. Disturbed paleosol sample was collected from the upper part of the profile (0-40 cm) immediately bellow Lower Albian limestone and air-dried afterwards. Undisturbed paleosol sample for thin section preparation was taken using 80 60 40 cm Kubiena box (10-18 cm). Particle size analysis was determined on the bulk sample after dispersion in water and separated by sedimentation in cylinder and quantitatively obtained after the appropriate settling time. The remaining portion in the cylinder was calculated as the 2- Major elements and several minor elements (Zr, Cr, Nb) were determined with an XRF spectrometer on glass discs. Trace elements were determined by ICP mass spectrometry following a multi-acid digestion. Sulfur and carbon were determined by the LECO analyser. -ray powder diffraction (XRD) using a Philips diffractometer (graphite monochromator, CuK radiation, proportional counter). The XRD patterns of bulk sample and non- taken after air- following treatments: (a) Mg-saturation, (b) K-saturation, (c) Mg-saturation and ethylene glycol solvation, (d) Mg-saturation and glycerol solvation, (e) K-saturation and DMSO solvation, and (f) heating for two hours at 550 C. The identification of clay minerals was 4

101 102 103 104 105 106 107 108 109 110 111 112 113 114 115 116 117 118 119 120 121 122 123 124 125 generally based on the methods outlined by Brown (12), Brindley & Brown (13), and Moore & Reynolds (14). The term illitic material was used as defin & Eberl (16). Thin section was prepared in the Thomas Beckmann laboratory (Schwülper Lagesbüttel, Germany) and analysed using Leica DM/LSP petrographic microscope with plane-polarized (ppl) and crossed-polarized light (xpl). An air-dried fragment of paleosol sample was gold-coated and analysed using a JEOL JSM-6510 LV Scanning Electron Microscope (SEM) fitted with an Oxford Instruments INCA system (EDAX). Selected ng SEM. Natively present cultivable bacteria in paleosol. The wet sample of paleosol was aseptically taken on May, 2013 in a sterile glass bottle and transferred to the laboratory within 4 h. The number of natively present cultivable bacteria was determined in fresh sample of paleosol in duplicate. A 1.0 g aliquot of paleosol was placed in a tube containing 9 ml of sterile 0.05M NaCl. Sample of paleosol was allowed to suspend in dark by shaking at 150 rpm for 24 h at 22 C. In this suspension the number of aerobically grown heterotrophic neutrophilic bacteria was determined as Colony Forming Units (CFU) on nutrient agar (Biolife) after 5 days of incubation at 22 C. For determination of anaerobically grown heterotrophic neutrophilic bacteria nutrient agar was cultivated in anaerobic conditions generated in Anaerocult A (Merck). The final ph value of the suspension of paleosol was measured using WTW 330 ph-meter. In order to determine the number of acidophilic bacteria in paleosol, a 1.0; 0.5 and 0.1 g aliquot of paleosol was placed in the tube with Leptospirillum HH medium (DSMZ medium 882) and incubated in dark for 7 days at 25 C. The presence of acidophilic bacteria was confirmed by scanning electron microscopy (Jeol JSM 5300, Japan) after the dehydration of samples in series of ethanol. Characterization of environmental A. baumannii isolate. The A. baumannii was isolated from aerobically cultivated nutrient agar plates. The isolate was firstly characterised 5

126 127 128 129 130 131 132 133 134 135 136 137 138 139 140 141 142 143 144 145 146 147 148 149 150 by routine bacteriological techniques: Gram staining, growth at 42ºC, catalase and oxidase reaction, and biochemical characteristics in API 20NE system (BioMerieux). Further identification was made using ATB 32GN and Vitek 2 systems (BioMerieux), as a standard procedure in microbiological laboratory and confirmed by the presence of OXA-69-like oxacillinase, a variant of OXA-51-type oxacillinase, specific for A. baumannii (17,18). - - -lactamase inhibitor combinations (ampicillin/sulbactam), trimethoprim/sulfamethoxazole, aminoglycosides (amikacin, gentamicin, tobramycin) and fluoroquinolones (ciprofloxacin, levofloxacin) were determined by disc-diffusion tests and confirmed by MICs using AST-XN05 and AST-N233 testing card for Vitek2 system, MICs values for colistin and ampicillin/sulbactam by E-tests (AB Biodisk), and interpreted according to the European Committee on Antimicrobial Susceptibility Testing criteria (19). The next step in characterisation of collected isolate of A. baumannii was genotyping using pulsed-field gel electrophoresis (PFGE) in order to investigate its similarity with clinical strains. We compared A. baumannii isolated from paleosol with three different isolates: a clinical isolate of A. baumannii from general hospital in Pula, Istria belonging to multicentre collection, and two clinical isolates belonging to European (international) clones I and II that are causing outbreaks in Croatia (20,21). General hospital in Pula is situated in the same area where our environmental isolate was found, so we supposed that it could be similar to the clinical isolate from this region. This clinical isolate was collected in 2009 during multicentric collection of carbapenem resistant isolates from southern Croatia and Istria. The PFGE analysis was performed according to Seifert et al. (22) with ApaI (New England Biolabs) as restriction enzyme, according to manufacturer s instructions. The resulting macrorestriction digests were electrophoresed in 1.4% agarose gel. PFGE fingerprinting patterns were analysed using Molecular Analyst Software for Finterprinting (Bio-Rad). Dice 6

151 152 153 154 155 156 157 158 159 160 161 162 163 164 165 166 167 168 169 170 171 172 173 174 175 similarity coefficients were calculated using pairwise comparison of the PFGE profiles, with optimisation of 1.5% and a position tolerance of 1.0%. Dendograms were created by using the unweighted pair group method with arithmetic averages (UPGMA). Biofilm formation of environmental A. baumannii isolate was compared to the multicentre collected clinical isolate. Overnight cultures were diluted to OD 600 of 0.1 in nutrient broth, distributed in polypropylene tubes and incubated at 37ºC for 24h without shaking. Biofilms were stained with 0.5% (w/v) crystal violet and quantified spectrophotometrically at 550 nm after solubilisation in 96% ethanol. Quantification of biofilm formation was performed in duplicate. OD 550 values of experimental tubes were subtracted from those of the negative control, which contained nutrient broth without bacteria. The production of extracellular substances in isolates was confirmed by alcian blue staining of 24 h old cultures cultivated in nutrient broth. Influence of low ph, desiccation and high temperature on environmental A. baumannii isolate. In order to test the influence of low ph on the A. baumannii isolate, the isolate was pre-grown on the nutrient agar (Biolife) for 16 h at 37±0.1 C. The biomass was then suspended in commercially available natural spring water of ph 8.29. The suspended biomass was inoculated in triplicate into tubes which contained 10 ml of: natural spring water; natural spring water adjusted to ph 3.40 with 1M HCl and natural spring water with addition of 1.0 g of air dried paleosol. Tubes were sealed and incubated at 22±0.1 C for 24 h with shaking at 150 rpm. The number of A. baumannii was determined in triplicate on nutrient agar after incubation at 37±0.1 C for 24 h. The numbers of CFU were logarithmically transformed and the survival was calculated as (log CFU 24h /log CFU c0 )*100. Statistical analyses were carried out using Statistica Software 10.0 (StatSoft, Tulsa, USA). The numbers of bacterial CFU were logarithmically transformed beforehand to normalize distribution and to equalize variances of the measured parameters. The comparisons between samples were 7

176 177 178 179 180 181 182 183 184 185 186 187 188 189 190 191 192 193 194 195 196 197 198 199 200 done using the one-way analysis of variance (ANOVA) and subsequently the post-hoc Duncan test was performed for the calculations concerning pair-wise comparisons. Statistical decisions were made at a significance level of p<0.05. The influence of desiccation and high temperature on A. baumannii isolate was done by drying of the original sample of paleosol at 50ºC for 72 h. A 1.0 g of dried paleosol was suspended in a tube containing 9 ml of sterile 0.05M NaCl and the numbers of A. baumannii were determined in triplicates on the nutrient agar after incubation at 42±0.1 C for 24 h. RESULTS Characterization of paleosol. Paleosol is dominantly composed of clay (<2 μm) with silt particles (2-63 μm) and sand particles (> 63μm) forming less than 5 wt.%. Clay minerals are main constituents of paleosol, while quartz, pyrite, gypsum and jarosite are present in silt and sand fractions and are minor mineral phases (Table 1). The main clay mineral is illitic material (Fig. 3) comprising 70 wt. % of clay fraction followed by illite/smectite mixed layer minerals (30 wt.%). Pyrite fills former roots, burrows and channels (Fig. 4), or is randomly distributed within clay matrix (Fig. 5). The high content of illitic material corresponds well with chemical data (Table 2). Namely, the K 2 O-content is around 5 wt.%, clearly indicating that the illitic material is dominant mineral phase in the paleosol. Based on infrared studies, Ottner et al. (10) found that the smectites in illite/smectite mixed layer minerals from clays in the Tri Jezerca quarry are aluminium-rich montmorillonites without iron substitution in the octahedral position. The obtained values of MgO (Table 2) match well with that. Durn et al. (11) concluded that the clay mineral composition of paleosol in the Tri Jezerca quarry clearly indicates the influence of both pedogenic and diagenetic processes. They concluded that paleosols were probably seasonally marshy soils or permanently waterlogged soils. High content of V, Mo, U, Ni and Zn (Table 3) are also in favour of acidic reductive pedogenic 8

201 202 203 204 205 206 207 208 209 210 211 212 213 214 215 216 217 218 219 220 221 222 223 224 paleoenvironment. Pyrite, which formed in paleosol both pedogenetically and diagenetically, is unstable mineral phase at the surface. The result of pyrite oxidation (Fig. 6), which lowers the ph of the soil, is the formation of secondary minerals gypsum (calcium originates from limestones below and/or above paleosol) and jarosite (hydrous sulphate of potassium and iron). Natively present cultivable bacteria in paleosol. In the fresh sample of paleosol no anaerobically grown heterotrophic neutrophilic bacteria were detected. Cultivation of the aerobically grown heterotrophic neutrophilic bacteria resulted in the growth of 80-120 CFU per 1 g of paleosol. The population was represented by only one morphological type of colonies. Grown colonies were picked and used for further characterization. The suspension of paleosol in 0.05M NaCl was acid with the final ph value of 2.55. In the fresh sample of paleosol acidophilic bacteria were detected by cultivation of 1.0 and 0.5 g of aliquots of paleosol in Leptospirillum HH medium, which is used for the cultivation of Acidithiobacillus ferrooxidans and Leptospirillum spp. (24). The rod shaped acidophilic bacteria in the extracellular polymeric matrix (Fig. 7) gave no results in the procedure of determination by MALDI-TOF mass spectrometry (Microflex LT MALDI-TOF MS, Bruker Daltonics). The total organic carbon (TOC) concentration in the examined sample of paleosol was 0.08 wt. % (Table 3). Due to the low TOC, the presence of acidophilic mesophilic heterotrophic bacteria (25) is unlikely. The acidophlic mesophilic chemolithotrophs A. ferrooxidans and L. ferrooxidans are the most significant bacteria involved in biological oxidation of pyrite and other sulphidic minerals. Acidophilic bacteria which oxidize reduced sulphur compounds but not ferrous ion, such as A. thiooxidans, support the leaching of minerals by production of sulphuric acid. Those bacteria degrade sulphide minerals by attachment onto minerals and enzymatic oxidation of ferrous ion or sulphide. The 9

225 226 227 228 229 230 231 232 233 234 235 236 237 238 239 240 241 242 243 244 245 246 247 248 extracellular polymers which are important in process of bacterial attachment may also play a role in leaching of sulphide minerals (26). Characterization of A. baumannii isolate. The colonies of aerobically grown heterotrophic neutrophilic bacteria were characterised by the routine bacteriological techniques (Gram staining, growth at 42ºC, catalase and oxidase reaction, biochemical characteristics in API 20NE system) as A. baumannii. One colony was chosen for subsequent analyses by ATB 32GN, Vitek 2 systems and the presence of OXA-69-like oxacillinase by PCR (18). The susceptibility testing by disc diffusion method and MICs values discovered the resistance pattern close to clinical isolates of A. baumannii from the most hospitals in Croatia according to the results of Public Health Collegium Committee for Antibiotic Resistance Surveillance (27). The isolate was susceptible to ampicillin/sulbactam (MIC 4 mg/l), amikacin (MIC < 2 mg/l), imipenem (MIC 0.5 mg/l), meropenem (MIC 0.5 mg/l), tobramycin (MIC < 1 mg/l), and colistin (MIC 0.5 mg/l), but resistant to gentamicin (MIC > 16 mg/l), trimethoprim/sulfamethoxazole (MIC 160 mg/l), ciprofloxacin (MIC > 4 mg/l) and levofloxacin (MIC 4 mg/l) interpreted according to EUCAST criteria (19). The result of molecular typing by PFGE confirmed that environmental isolate of A. baumannii showed 87% of similarity with the clinical isolate obtained from general hospital in Pula (Fig 8) and represent a cluster inside the European clone I. European clone I has been recognized as a cause of of nosocomial infection in Croatia for over 10 years, and for a long time it has been the dominant clone causing hospital outbreaks (20). However, the resistance pattern to carbepenems of clinical isolate in Pula display reduced susceptibility to imipenem and meropenem (MICs 8-16 mg/l) carrying a blaoxa-69-like gene associated with ISAba1 which is well described mechanism of carbapenem resistance in Croatia in the last decade (20,21). 10

249 250 251 252 253 254 255 256 257 258 259 260 261 262 263 264 265 266 267 268 269 270 271 272 273 Quantification of biofilm formation resulted in OD 550 values of 0.587±0.100 for environmental isolate and 0.022±0.007 for clinical isolate of A. baumannii. Since the difference of OD 550 and negative control for environmental isolate was 7.4±0.9 and for clinical isolate 1.2±0.1, the environmental isolate could be interpreted as biofilm-forming while clinical isolate biofilm-negative strain. The alcian blue staining confirmed the presence of thick layer of extracellular substances in environmental isolate of A. baumannii, which were much more abundant than in clinical isolate (Fig 9). The extracellular polysaccharides are important in attachment of bacteria onto particles and protect bacterial cells in unfavourable environmental conditions (28). Influence of low ph, desiccation and high temperature on A. baumannii isolate. The influence of low ph (initially adjusted or generated after addition of paleosol) on the environmental isolate of A. baumannii is shown in Table 4. The isolate showed 100% survival in the natural spring water of neutral ph. Survival of the isolate in the acid spring water was not significantly different than the survival in the acid spring water generated by paleosol. This suggests that low ph was the major reason for the reduction of number of A. baumannii in contact with paleosol. The relatively high content of potentially toxic heavy metals in paleosol (Table 3) was not important for the survival of A. baumannii. The survival of A. baumannii in contact with paleosol (20-100 CFU/g) was good enough to support the findings of 80-120 CFU/g from fresh paleosol. In the sample of paleosol which was dried at 50ºC for 72 h a 60-100 CFU/g of aerobically grown heterotrophic neutrophilic bacteria were detected. The population was again represented by only one morphological type of colonies. The characterization of the colony (performed as described for the fresh sample) showed that the isolated bacterium was A. baumannii. Obviously, the desiccation and high temperature showed no significantly negative influence on the survival of the population of A. baumannii in fresh paleosol. 11

274 275 276 277 278 279 280 281 282 283 284 285 286 287 288 289 290 291 292 293 294 295 296 297 298 DISCUSSION Reports regarding the occurrence of viable clinically important multi-drug resistant Acinetobacter sp. in nature are scarce. While it is certainly true that A. baumannii can be isolated from patients and hospital environmental sources during outbreaks, this species has no known natural habitat outside the hospital (29). Multiresistant isolates of A. baumannii were found in the wastewater from hospitals in Brazil (30). A. baumannii was also isolated from hospital solid waste (31). In the study on occurrence of A. baumannii in the soil, the screening of 49 soil samples resulted in only one soil sample from which Acinetobacter sp. was recovered (5). This isolate was not genetically related to any of the clinical isolates. Such investigation may underestimate the significance of soil as a source of clinically related Acinetobacter sp. Namely, the procedure of the isolation employed the placement of swab in soil sample, after which the swab was streaked across the agar plate. By such procedure a small quantity of soil is analysed. The conditions and storage duration of archived soil samples may result in limited recovery of environmental isolates. The analysis of large quantity of fresh soil is promising in the successful isolation of clinically related Acinetobacter sp. from environment. Our environmental isolate of A. baumannii showed similarity with the compared clinical isolate in susceptibility pattern to ampicillin/sulbactam, colistin, amikacin and tobramycin. However, our environmental isolate was susceptible to carbapenems (both imipenem and meropenem), which is not unexpected knowing the main basis of carbapenem resistance till 2009 in Croatia (20). According to the published data and surveillance follow up in the last ten years the main mechanism of carbapenem resistance was the presence of insertion sequence ISAba1 upstream of bla OXA 51/69 like gene (20,32). Since we are talking about a mobile genetic element that gets full expressions in the conditions of excessive consumption 12

299 300 301 302 303 304 305 306 307 308 309 310 311 312 313 314 315 316 317 318 319 320 321 322 323 (overuse) of antibiotics (in this case, carbapenems), this mechanism of resistance is not expected to be found in environmental isolate. The nearest medical institutions to the Tri jezerca quarry are situated at the seaside, more than 10 km away. Therefore, it is hard to believe that the A. baumannii isolated from paleosol origins from the hospital wastewaters (30). The probable source of A. baumannii in the upper part of the paleosol profile (0-40 cm) is the illegally disposed waste of different origin (31) situated in abandoned quarry where investigated paleosol is situated. The bacteria could have been leached from waste by storm water, and infiltrated in paleosol. In the wet environment Acinetobacter sp. have potential to survive for the prolonged period of time. At the relative humidity of 31% strains of A. baumannii survived up to 36 days (33,34). On dry formica surface A. calcoaceticus survived up to 13 days (35). 39 of 118 of human skin isolates of Acinetobacter sp. tolerated the temperature of 50ºC (36). Acinetobacter sp. grow well in the ph range 5-8 (7). The planktonic A. junii did not survive the ph 3 during 24 h of contact, while A. junii immobilized onto zeolite particles successfully survived (28). There are no literature reports on the survival of Acinetobacter sp. on acid minerals. In the paleosol A. baumannii are immobilized onto soil particles. The immobilized cells are protected by clay particles in soil which can enhance the survival of bacteria when compared to planktonic cells. The extracellular polymeric substances of A. baumannii may have the additional protective role in unfavourable environmental conditions (low ph, desiccation and high temperature). The input of A. baumannii in the reported paleosol was probably much greater than detected 80-120 CFU/g of paleosol. Despite of wet soil, only a part of the bacterial population survived due to the low ph of paleosol. The survival of A. baumannii in the neutral or alkali soil would be much greater. The finding of clinically related A. baumannii in paleosol suggests that illegal landfill could be the source of multi-drug resistant bacteria in nature. 13

324 325 326 327 Therefore, illegal landfills represent threat to the environment. Detection of this isolate and their ability to survive in paleosol opens up new options in finding the spread of these pathogens outside the hospital environment and represents a potential new source of infection, even in immunocompetent hosts. 328 329 330 331 332 333 334 335 336 337 338 339 340 341 342 343 344 345 346 347 348 ACKNOWLEDGEMENTS This research was supported by the Ministry of Science, Education and Sports of the Republic of Croatia (project nos. 119-1191155-1203 and 195-1953068-2704). We thank to all Croatian collaborative centers for collecting clinical isolates of A. baumannii for multcenters investigations, especially Nada Barisic and Mirna Vranic-Ladavac (GHP). REFERENCES 1. Towner KJ. 2009. Acinetobacter: an old friend, but a new enemy. J. Hosp. Infect. 73:355-363. 2. Roca I, Espinal P, Vila-Farres X, Vila J. 2012. The Acinetobacter baumannii Oxymoron: commensal hospital dweller turned pan-drug-resistant menace. Front Microbiol. 3:1-30. 3. Camp C, Tatum OL. 2010. A review of Acinetobacter baumannii as a highly successful pathogen in times of war. Lab. Med. 41:649-657. 4. Gilbert DN, Guidos RJ, Boucher HW, Talbot GH, Spellberg B, Edwards JE, Scheld WM, Bradley JS, Bartlett JG. 2010. The 10 x 20 Initiative: Pursuing a Global Commitment to Develop 10 New Antibacterial Drugs by 2020. Clin. Infect. Dis. 50:1081-1083. 5. Scott P, Deye G, Srinivasan A, Murray C, Moran K, Hulten E, Fishbain J, Craft D, Riddell S, Lindler L, Mancuso J, Milstrey E, Bautista CT, Patel J, Ewell A, Hamilton 14

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444 445 446 447 448 449 450 451 452 453 FIG 4 Root remains, burrows and channels, mainly filled with pyrite (upper part of profile, 10-18 cm); section represent dimension of 3.3 mm. FIG 5 SEM microphotograph of pyrites in clay matrix. FIG 6 SEM microphotograph of weathered pyrite. FIG 7 Acidophilic bacteria on the surface of paleosol surrounded with the network of extracellular polymeric matrix. Arrows indicate the bacterial cells. FIG 8 Dendogram based on ApaI-digested DNA from different isolates of A. baumannii. FIG 9 Thick layer of extracellular substances (blue) with embedded cells (red) of environmental isolate (A) and less abundant extracellular substances in clinical isolate of A. baumannii (B). Downloaded from http://aem.asm.org/ on March 21, 2019 by guest 19

TABLE 1 Mineral composition (in wt. %) of the bulk paleosol sample (P1). Phyllosilicates = Phyllosilicates and amorphous inorganic compound. Sample Quartz Pyrite Gypsum Jarosite Phyllosilicates P1 1 2 1 1 95 TABLE 2 Chemical composition (in wt. %) of the bulk paleosol sample (P1). Color (dry) after Munsell Soil Color Charts (23). SiO 2 to LOI in wt. %. Sample Color SiO 2 TiO 2 Al 2 O 3 Fe 2 O 3 MgO CaO Na 2 OK 2 OP 2 O 5 LOI Sum P1 5BG4/1 51.42 1.26 24.30 5.59 2.69 1.09 0.23 5.14 0.02 7.65 99.39 TABLE 3 Content of sulfur (S), carbon (C) and trace elements in the bulk sample of paleosol (P1). S and C in wt. %, Ba to Zr in ppm. Sample S C Ba Co Cr Cu Ga Mo Nb Ni Pb Rb Sr Th U V Y Zn Zr P1 1.5 0.08 248 15 162 25 30 92 19 187 27 257 87 22 36 469 14 142 271

TABLE 4 Survival of A. baumannii after 24 h of contact in natural spring water (positive control); natural spring water adjusted to ph 3.40; and natural spring water with addition of 1.0 g/10 ml of paleosol. c 0 (10 7 CFU/mL) = 5.14±1.08; survival was calculated as (log CFU 24h /log CFU c0 )*100; *significantly lower as compared to positive control; no statistical difference was found between spring water of ph 3.40 and spring water with paleosol. Parameter Positive control Spring water of ph 3.40 Spring water with paleosol ph start 8.29±0.02 3.40±0.02 8.29±0.02 ph final 7.89±0.12 3.53±0.09 3.37±0.03 Survival (%) 100.9±1.2 8.9±4.6* 10.5±3.7* Downloaded from http://aem.asm.org/ on March 21, 2019 by guest