Carbapenem-resistant isolates of Acinetobacter baumannii in a municipal wastewater treatment plant , Croatia , 2014

J Hrenovic 1 , I Goic-Barisic 2 , S Kazazic 3 , A Kovacic 4 , M Ganjto 5 , M Tonkic 2 1. University of Zagreb, Faculty of Science, Department of Biology, Zagreb, Croatia 2. University Hospital Centre Split, Department of Clinical Microbiology and University of Split School of Medicine, Split, Croatia 3. Ruđer Boskovic Institute, Division of Physical Chemistry, Zagreb, Croatia 4. Institute of Public Health of Split and Dalmatia County, Split, Croatia 5. Zagreb Wastewater Management and Operation Ltd., Zagreb, Croatia


Introduction
Over the last decade, hospital-acquired infections due to Acinetobacter baumannii have increased dramatically worldwide including in Croatia [1,2].Difficulties caused by this pathogenin the hospital setting are exacerbated by its abilities to form biofilms on abiotic or biotic surfaces and to cope with different environmental conditions, including desiccation and disinfectants [3][4][5].Non-susceptibility to commonly used antimicrobials [6] has also been observed, with carbapenem resistance becoming a global problem since 2000 [7,8].In Croatia, proportions of carbapenem-resistant A. baumannii strains in clinical samples increased drastically from 2008 to 2012 (10% to 73% of isolates respectively), with some individual hospitals recording a rate of 90% [2,9].
How A. baumannii is introduced into the hospital environment remains incompletely understood [5,15].Acute community-acquired human infections have been reported [16] and suggest a source of the pathogen outside of the hospital.Even if such infections are mainly reported from tropical and subtropical areas and represent only a minor proportion of all A. baumannii infections worldwide, community-acquired infections underline the importance of searching for possible environmental reservoirs of the pathogen and their potential consequences.In nature, multidrug-resistant (MDR) A. baumannii strains have been isolated from hospital wastewaters [17,18].Aside from reports of A. baumannii in wastewaters close to hospitals, only few studies report detection elsewhere in the environment [19,20].
The aim of this study was to screen municipal wastewater for the presence of viable A. baumannii, characterise the recovered strains as well as investigate their potential for survival in the environment after passage through the wastewater treatment process.

Sampling and characterisation of wastewater
Wastewater was collected at the largest Croatian wastewater treatment plant of Zagreb.This plant (capacity 1,200,000 population equivalents) is designed for the secondary treatment of municipal wastewater, which originates from a combined sewage system of domestic, hospital, industrial and storm water.The central treatment plant receives wastewater from all nine clinical hospitals of Zagreb.In accordance with the national legislation, sanitary hospital wastewaters are released into the sewage system without pre-treatment, and only radioactive wastewaters and infective solid waste are disposed of separately.The sewage system of Zagreb is an underground network of sewer with different retention time of wastewater depending on the inhabitants' behaviours and stormwater runoff.
Composite 24h samples of influent and effluent wastewater were collected in June and October 2014 and April and October 2014, respectively.The physicochemical parameters of water samples were measured according to the Standard Methods for Examination of Water and Wastewater [21].Temperature was measured online and average values of composite samples were calculated.

Bacteriological analyses
For bacteriological analyses, wastewater was aseptically sampled in sterile 1L glass bottles and transferred to the laboratory within 1h.The wastewater samples were concentrated on sterile membrane filters of pore size 0.45µm in triplicate both before and after dilution in sterile peptone water.
Intestinal enterococci were determined according to HRN ISO 7899-2 [22].Membrane filters were incubated on Slanetz Bartley agar (Biolife) at 37 °C for 72h and subsequent confirmation of intestinal enterococci (i.e.colonies with dark brown halo) was done on bile esculin azide agar (Sigma-Aldrich) after incubation at 44 °C for 4h.The isolates' names according to their origin (influent or effluent wastewater), as well as the carbapenemase they harboured and date of collection are shown on the right hand side of the dendogram.
a A total of 21 A. baumannii isolates were obtained throughout the study but the ApaI digestion failed for one, hence 20 isolates were further analysed by pulsed-field gel electrophoresis and presented in the dendogram.
Carbapenem-resistant bacteria were determined on CHROMagar Acinetobacter supplemented with CR102 (CHROMagar), which allows the growth of carbapenemresistant isolates after incubation at 42 °C for 48h.
The numbers of intestinal enterococci, aerobically grown total heterotrophic bacteria, and carbapenemresistant bacteria were determined as colony forming units (CFU), logarithmically transformed, and expressed as log CFU per 1 mL of water.The prevalence of carbapenem-resistant bacteria among total heterotrophic bacteria was calculated.

Isolation and characterisation of Acinetobacter baumannii isolates
There is no simple protocol for the isolation of viable A. baumannii from environmental samples.A. baumannii is usually overgrown with accompanying flora even on selective and differential media.For the iso-  Pure cultures of presumptive A. baumannii grown at 42 °C on Nutrient agar were firstly characterised by routine bacteriological techniques to assess the following characteristics: Gram negative coccobacilli, negative oxidase, positive catalase reaction, no reaction on the Kligler Iron Agar (Biolife).Further identification of presumptive A. baumannii was carried out by ATB 32GN and Vitek 2 systems (BioMerieux) [23,24].
Matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) which was shown to be a reliable method for rapid identification of A. baumannii [25,26] was used for confirmation on cell extracts according to Sousa et al. [27].In brief, overnight cultures pre-grown on Nutrient agar were suspended in 70% ethanol.After centrifugation and removal of the supernatant, cultures were extracted with 70% formic acid and an equal volume of acetonitrile.The suspension was centrifuged and cell extracts were spotted onto a MALDI target plate and air dried at room temperature.Subsequently, the sample was overlaid with a saturated solution of α-cyano-4-hydroxycinnamic acid in 50% acetonitrile and 2.5% trifluoroacetic acid as a matrix, dried, and subjected to analysis.Spectra were obtained by using Microflex LT (Bruker Daltonics) in the linear positive mode in the range 2-20 kDa.Each recorded spectrum is the result of six series of 40 single laser shots in different locations.Identification of isolates was performed in technical triplicate and biological duplicate.Recorded mass spectra were processed with the MALDI Biotyper 3.0 software package (Bruker Daltonics) using standard settings.
The genetic relatedness of A. baumannii isolates from wastewater to clinical isolate was assessed by pulsedfield gel electrophoresis (PFGE).Representatives of A. baumannii belonging to the international clonal lineage I (RUH 2037) and II (RUH 134), which have been confirmed as two dominant clones causing outbreaks in Croatia since 2002 and which are still present in Croatian hospitals [8,9,[12][13][14], were used as PFGE reference strains.PFGE analysis was performed using CHEF-DRII/III system (Bio-Rad) with ApaI (New England BioLabs) as restriction enzyme.The images of gelelectrophoresed restriction products were processed using Gel-Doc 1000 system (Bio-Rad) and Compar software.PFGE profiles were analysed and compared using Molecular Analyst Software for Fingerprinting (Bio-Rad).Matching and dendogram were performed with the unweighted pair group method with arithmetic averages (UPGMA) analysis [28,29] using Dice similarity coefficient with optimisation and a position tolerance of 1.0%.The isolates were classified into clusters based on their genetic similarity (cut-off of ≥ 90%).

Antibiotic resistance of Acinetobacter baumannii isolates
Susceptibility to β-lactams (imipenem, meropenem), fluoroquinolones (ciprofloxacin, levofloxacin), aminoglycosides (amikacin, gentamicin, tobramycin), and trimethoprim/sulfamethoxazole were determined by disc-diffusion tests.The minimum inhibitory concentration (MIC) values were confirmed using AST-XN05 and AST-N233 testing card for Vitek2 system or using E-tests (AB Biodisk) for colistin (which was also included for susceptibility testing), and interpreted according to the European Committee on Antimicrobial Susceptibility Testing criteria [30].
The carbapenemase-induced carbapenem degradation was confirmed by MALDI-TOF MS as described by Burckhardt and Zimmermann [31] and Hrabak et al. [32], with slight modifications.In brief, a loopful of overnight bacterial culture was added to 0.5mL of 0.2mM meropenem trihydrate (Tokyo Chemical Industry Co., Ltd.) in 0.05M NaCl.The solutions were incubated at 37 °C for 2.5h with stirring at 150rpm.After centrifugation, the supernatants were spotted onto a MALDI-TOF MS targets by a sandwich method using 2,5-dihydroxybenzoic acid (DHB) as a matrix, and subjected to analysis.A carbapenem-resistant clinical isolate of A. baumannii carrying the OXA-72 carbapenemase [13] was used as a positive control, while a meropenem solution without addition of bacteria served as the negative control.
The presence of the genes of bla OXA lineage which encode OXA-type carbapenemases was checked in 14 isolates of A. baumannii, selected on the basis of PFGE pattern and antibiotic susceptibility profiles.Multiplex polymerase chain reaction (PCR) with specific respective primers was used to amplify bla OXA-51-like , bla OXA- 40-like , bla OXA-23-like and bla OXA-58-like genes, according to Woodford et al. [33].CFU: colony forming units.

Survival of Acinetobacter baumannii in effluent wastewater
Survival of six isolates of A. baumannii (isolates EF1-6) recovered from effluent wastewater was monitored in the sterilised effluent wastewater collected on 28

Physical, chemical and bacteriological characteristics of wastewater
Temperatures of pH-neutral wastewater were relatively constant (Table 1).The physico-chemical characteristics of wastewater slightly varied on two sampling occasions of influent or effluent.Most evident however, were variations in inflow and outflow and concentration of dissolved oxygen where the influent in June was anoxic (0.09 mg dissolved O 2 /L).Both in June and in October, the ratio of five-day biochemical oxygen demand (BOD 5 ) to chemical oxygen demand (COD) in the influent was 0.5, which is considered high (0.3-0.6) and suggests nutrient rich biodegradable influent wastewater [34].Concentrations of nutrients in effluent wastewater were however in compliance with the national emission standards [35].
National emission standards do not require the monitoring of microorganisms at a described type of wastewater treatment plant.High levels (> 1.8 log CFU/mL) of intestinal enterococci (Table 1) that are reliable indicators of faecal pollution [36] were found in the influent and effluent water at each sampling occasion.The prevalence of carbapenem-resistant bacteria among total heterotrophic bacteria ranged from 34 to 57% in influent and 22 to 29% in effluent.

Isolation and characterisation of Acinetobacter baumannii isolates
For the whole study, a total of 21 A. baumannii were isolated; 15 from influent and six from effluent wastewater.Colonies of A. baumannii were circular, convex, smooth, red with a paler central area.All isolates of A. An influent isolate named IN19 appeared resistant to ApaI digestion and was therefore not subjected to subsequent PFGE cluster analysis.For the remaining A. baumannii isolates recovered from wastewater, none were similar to isolates belonging to the international clonal lineage I or II based on the PFGE dendrogram (Figure 1).Three effluent wastewater samples collected on 16 April clustered together as well as all but one of the influent samples from 11 June, and all but one of the influent samples from 28 October.The effluent samples from 28 October, however, showed the absence of clusterisation.

Isolates IN4 and IN5, IN13 and IN17, IN14 and IN16
showed pairwise 100% identical PFGE profile.These PFGE identical pairs were respectively collected on the same date, all from influent water.

Antibiotic resistance of Acinetobacter baumannii isolates
According to MIC values of tested antibiotics ( of incubation (Figure 2).All isolates from wastewater degraded the meropenem within 2.5h.
Selected types of spectra are shown in Figure 2. The positive control carbapenem-resistant clinical isolate of A. baumannii yielded a typical spectrum of complete meropenem degradation with characteristic peaks at m/z 379, 401, 423, and 445.Three isolates (IN4, IN5, IN8) of A. baumannii from wastewater degraded the meropenem as efficiently as the clinical isolate, as represented by the spectra of isolate IN4 in Figure 2. In the spectra of the remaining A. baumannii isolates recovered from wastewater in this study, residues of meropenem sodium and disodium salts (m/z 405 and 427) were visible in addition to the meropenem degradation products.This type of meropenem degradation is represented by the spectra of isolate EF1 in Figure 2 and suggests that such wastewater isolates degraded meropenem slower than the clinical isolate.A contact time of more than 2.5h was needed for these environmental isolates to fully degrade 0.2mM of meropenem.These isolates degraded the meropenem molecule first, followed by the degradation of meropenem sodium and disodium salts.
Monitoring of the meropenem degradation by MALDI-TOF MS does not provide detection of the type of carbapenemase carried nor detection of other carbapenem-resistance mechanisms [31].The presence of genes of bla OXA lineage which encode OXA-type carbapenemases was further confirmed by PCR.All of 14 tested A. baumannii isolates harboured the constitutive bla OXA-51-like gene.Among these, 10 isolates had the acquired bla OXA-23-like , three the acquired bla OXA-40-like genes and one isolate did not have any acquired bla OXA gene (Table 2).
For the three pairs of identical isolates by PFGE, the bla OXA genes in two pairs were only determined for one isolate respectively.For the remaining pair comprising IN14 and IN16, the acquired bla OXA-23-like gene was confirmed for both isolates in the pair (Table 2 and Figure 1).The bla OXA-23-like gene was moreover found in two tested isolates from the three effluent samples of 16 April, which clustered together by PFGE.It was also present in the four of six tested influent water sample isolates from 28 October, which together formed a separate in PFGE.The four remaining isolates with bla OXA-23- like gene did not cluster together by PFGE and included three effluent isolates from 28 October and one influent isolate from 11 June.Although influent samples from 11 June formed a larger PFGE group, this group contained a sub-cluster with two isolates harbouring bla OXA-40-like genes.The third isolate with bla OXA-40-like gene was from an influent water sample collected on 28 October.

Survival of Acinetobacter baumannii in effluent wastewater
Six isolates of A. baumannii recovered from effluent wastewater showed excellent survival in the autoclaved effluent wastewater from 28 October (Figure 3).The low concentration of nutrients in this effluent wastewater was sufficient to support the multiplication of bacteria.Multiplication of isolates occurred after one day of incubation, and log phase of growth was evident up to nine days of incubation.The stationary phase of growth was continued up to 30 days, after which a slight decrease of log CFU was observed up to 50 days of incubation.After 50 days of monitoring the numbers (log CFU) of A. baumannii isolates were 4.4 to 15.7% higher than initial numbers.

Discussion
In this investigation, wastewater collected at a water treatment plant in Zagreb in April, June and October 2014 tested positive for A. baumannii at each occasion.A previous study in 2002 [37] reported the presence of eight phosphate-accumulating A. baumannii strains in the activated sludge of a municipal wastewater treatment plant in Izmir, Turkey.The strains in the latter study were however only characterised biochemically (API 20NE identification kit) so they could have belonged to other species of the genus Acinetobacter considered important for enhanced biological phosphate removal from wastewaters [24,27,38].In contrast, none of the A. baumannii isolates in the current study were able to accumulate intracellular polyphosphate granule as confirmed by Neisser staining (data not shown).
The prevalences of carbapenem-resistant bacteria among total heterotrophic bacteria, which were estimated each time the water was sampled, ranged from 34 to 57% in influent water and 22 to 29% in effluent water.As carbapenems are heavily used in clinical environments, such prevalences suggest hospital wastewater in the influent water and bacteria of clinical origin in the effluent.The finding of OXA-40-like carbapenemase in three of 14 A. baumannii isolates recovered from wastewater strengthens this hypothesis.Indeed, OXA-40-like carbapenemases constitute the dominant mechanism of carbapenem resistance in clinical isolates of A. baumannii from most Croatian hospitals [9,13,14].In addition to this, a number of strains isolated from wastewater degraded meropenem in the same way as a control clinical isolate.The dominance (10 of 14) of wastewater isolates harbouring the bla OXA-23-like gene was nevertheless unexpected, since the clinical isolates of A. baumannii which produce OXA-23-like carbapenemases have only been observed sporadically in the country [14].It is possible that isolates carrying the bla OXA-23-like gene are more resistant to adverse environmental conditions in sewage as well as in the wastewater treatment process, thus surviving longer, but this needs to be further examined.
In spite of this, all wastewater A. baumannii isolates recovered in this study were MDR and showed comparable levels of antibiotic resistance to clinical A. baumannii isolates in Croatia [12].As environmental bacterial isolates are generally more susceptible to antibiotics than clinical isolates [39], these results also point to hospitals as the likely source of MDR A. baumannii in the wastewaters considered.In a preliminary screening, we confirmed the presence of MDR A. baumannii in the wastewater of one hospital in Zagreb (data not shown).The observation of MDR A. baumannii in hospital wastewaters has also been previously reported in Brazil and China [17,18].
High numbers of intestinal enterococci that are reliable indicators of faecal pollution [36] were also found in the wastewater together with A. baumannii, suggesting the presence of sanitary water and bacteria of faecal origin.MDR A. baumannii has been found in digestive tracts of hospitalised patients [40] as well as in animal faeces and urine [41].The digestive tracts of colonised patients and animals could be important epidemiological reservoirs of MDR A. baumannii, from which bacteria could migrate through wastewater into the natural environment.The wastewater from veterinary clinics [41] and stormwater, which could leach the bacteria from solid waste [20] may also represent a source of A. baumannii.Multilocus sequence typing (MLST) of housekeeping genes should be further performed in order to assess the genetic relatedness of A. baumannii isolates from municipal wastewater and clinical isolates, as well as isolates from hospital wastewaters in Zagreb.
When allochthonous bacterial species come in the environment, some abiotic and biotic ecological factors have been shown to determine their survival and multiplication.Little is known however about the influence of environmental factors on A. baumannii strains which are significant for public health in wastewater.Six isolates of A. baumannii recovered from effluent wastewater multiplied and survived in the effluent wastewater for up to 50 days of monitoring.The average annual water flow velocity of the Sava River, which receives the effluent wastewater, is 0.75m/s, and 12 days are needed for the water mass to travel from the location of effluent discharge to the firth of the Sava into the Danube River.Therefore, the viable MDR A. baumannii could possibly spread through the natural water bodies or accumulate in river sediments.A separate study finding a MDR A. baumannii isolate related to a clinical isolate in the Seine River in France further supports this possibility [19].
Zhang et al. [39] reported that the tertiary wastewater treatment process contributed to the selective increase of MDR Acinetobacter spp. in final effluent and natural recipient.Lower numbers of carbapenem-resistant bacteria together with lower prevalence of such bacteria among total heterotrophic bacteria in effluent than in influent wastewater does not support this in the case of our secondary wastewater treatment process.Higher numbers of viable MDR A. baumannii isolated per sampling occasion from influent than from effluent wastewater rather suggest a beneficial role of the secondary wastewater treatment process on the distribution of clinically important bacteria in the environment.
However, certain numbers of carbapenem-resistant bacteria and A. baumannii are released after the secondary treatment through the effluent wastewater into the natural recipient, which is the Sava River.The presence of viable MDR A. baumannii in both raw and treated municipal wastewater poses a serious concern about the spread of this emerging pathogen in nature.Disinfection of effluent water has been suggested as a promising tool for prevention of the accumulation of MDR bacteria in waterbodies [42].However, currently the standards for discharge of treated municipal wastewater do not prescribe the elimination of MDR bacteria.Furthermore, carbapenem-resistant MDR A. baumannii were isolated from hospital wastewaters both prior and after disinfection by chlorination [18].Genes encoding New Delhi metallo-beta-lactamase-1 (NDM-1) were present in domestic and industrial wastewater and each stage of the wastewater treatment process, including the chlorinated effluent [43].Although the viable bacteria were not determined, these NDM-1 genes were 99.8% identical to the NDM-1 genes carried by A. baumannii.The findings of MDR A. baumannii and NDM-1 genes after the process of chlorination suggest that conventional disinfection of effluent may not be the best strategy for mitigating the propagation of A. baumannii in the environment.Other disinfection techniques or barrier approaches at the source of contamination may be a more promising approach.
Tracking the source and further dispersion of A. baumannii contamination is needed to initiate any response plan for its control in the environment.The isolates of A. baumannii recovered from the environment should be compared with hospital, veterinary/livestock and community-acquired strains by using additional types of molecular assays such as whole genome sequencing [16], whereby there may be identifiable differences between these strains.Larger screening and epidemiological studies should also be performed to investigate the impact of human waste on the spread of MDR A. baumannii in the natural environment.In the current era when new antimicrobials for MDR Gram-negative microorganisms are scarce, it is most important to understand the epidemiology of this human pathogen.The results of this study represent a step forward in this direction.
colonies of A. baumannii were isolated from plates inoculated with 0.01-0.1mL of influent water and 0.1-1.0mL of effluent water.Presumptive colonies of A. baumannii were recultivated (42 °C for 24h) on the same selective plates and then on Nutrient agar.

Table 1
Physical, chemical and bacteriological characteristics of influent and effluent wastewater from a secondary water treatment plant, by sampling date, Zagreb, Croatia, 2014 CFU: colony forming unit; COD: chemical oxygen demand; BOD 5 : biochemical oxygen demand in five days; SD: standard deviation.aPrevalence of carbapenem-resistant bacteria among total heterotrophic bacteria was calculated as (log CFU/mL carbapenem resistant /log CFU/mL heterotrophic ) x 100.baumannii from wastewater gave reliable score values (range: 2.048-2.409)when compared with A. baumannii strains in MALDI Biotyper database.All isolates were also determined by Vitek 2 system as A. calcoaceticus-baumannii complex.

Table 2
The carbapenemase-induced carbapenem degradation by A. baumannii isolates from wastewater was confirmed by MALDI-TOF MS.No spontaneous degradation of meropenem was observed and characteristic peaks (m/z 383, 405, 427) were evident after 2.5h

Table 2
Minimum inhibition concentration values of tested antibiotics and presence of acquired genes of bla OXA lineage a in isolates of Acinetobacter baumannii, Zagreb, Croatia, 2014 AMK: amikacin; CIP: ciprofloxacin; CST: colistin; GEN: gentamicin; IPM: imipenem; LVX: levofloxacin; MEM: meropenem; SXT: trimethoprimsulfamethoxazole; TOB: tobramycin; R: resistant according to European Committee on Antimicrobial Susceptibility Testing criteria.a The constitutive gene bla OXA-51-like was confirmed in all isolates.b No acquired bla OXA-like gene.