Analysis of migration of pathogenic drug-resistant bacteria to soils and groundwater after fertilization with sewage sludge.

The paper discusses the analysis of the effect of using sewage sludge for fertilization on the level of soil and groundwater contamination with drug-resistant bacteria. Other sanitary contaminants in these environments were also analysed. Composted sewage sludge was introduced into the sandy soil over a period of 6 months. The examinations were conducted under conditions of a lysimetric experiment with the possibility of collecting soil leachates (in natural conditions). The following doses of sewage sludge were used: 0, 10, 20, 30 and 40 t/ha calculated per experimental object containing 10 kg of sandy soil. The research were carried out within the time frame of one year. Dactylis glomerata grass was grown on the fertilized soils. In soils and leachates from soils (which may have polluted groundwater) collected from fertilized experimental objects, the sanitary condition and quantity of drug-resistant bacteria (mainly from the families Enterobacteriaceae and Enterococcus) were analysed one year after fertilization. Their drug resistance to selected antibiotics was also analysed based on current recommendations. The study showed that fertilization with sewage sludge (even after stabilization and hygienization) results in contamination of soil and infiltrating waters with many species of drug-resistant pathogenic bacteria. The lowest level of contamination of soil and water environment was found after the application of sewage sludge at a dose of 10 t/ha. The isolated drug-resistant strains of intestinal bacteria were less sensitive to older generations of antibiotics including cefazolin, ampicillin, and co-amoxiclav.

1. Introduction

The content of various drugs, including antibiotics, is often determined in the soil and aquatic environments. They are detected in surface water, groundwater and even in water purified and intended for consumption [1]. At the same time, new drug-resistant bacteria are often identified. They can also be found more and more often in food [2, 3]. The sources of these pollutants are mainly wastewater from pharmaceutical industrial plants and hospitals [4]. Hospital sewage is particularly dangerous [5, 6]. The sources of contamination also include domestic sewage, sewage sludge and waste from agriculture and animal husbandry [7-11].

In wastewater treatment plants, antibiotics are removed to an insufficient level [12, 13]. The consequence is the migration of antibiotics and their toxic metabolites to the aquatic and soil environment. Wastewater can be a source of contamination of soil, plants and water, including rivers and seas [14], with bacteria resistant to many drugs. Studies have documented the contamination of fish and shrimps with drug-resistant bacteria from the Enterobacteriaceae family [15].

Fertilization of soils with organic fertilizers and waste, including sewage sludge, may cause the spread of pathogenic microorganisms in the environment. There are reports on the presence of drug-resistant bacteria in sludge in the literature concerning sludge management [12, 16]. In both sludge and a conventional organic fertilizer provided by manure, numerous pathogenic forms have been found while they acquired or developed resistance mechanisms with respect to a number of antibiotics available in health care [17-19]. Particularly risky is the possible migration of these harmful forms with precipitation and water infiltrated to groundwater and further to drinking water intakes.

Sanitary cleanness of water, including groundwater, is one of the most important components of contemporary water management. However, many potentially hazardous microorganisms are often found in water [20].

Many studies also highlight the risk of environmental migration and the emergence of pathogenic forms of drug-resistant bacteria in water. One of the ways of contaminating groundwater can be organic waste fertilization, including the use of sewage sludge (which is a reservoir of various microbial species). It is therefore important to determine the risk of the natural use of this waste. The authors undertook a study to determine the effect of sewage sludge application at different doses on the sanitary status of soils and migration of pathogenic microorganisms, including Enterobacteriaceae and Enterococcus family bacteria, to fertilized soils and groundwater.

The presence of sanitary indicators, intestinal bacteria and enterococci was determined in the analyzed samples of sewage sludge used for fertilization, fertilized soils and waters infiltrating the soils. The authors analyzed the effect of using sewage sludge for fertilization (in doses 0, 10, 20, 30 and 40 t/ha) on the degree of soil and groundwater contamination with drug-resistant bacteria.

Studies have shown that there is a threat of migration of pathogenic bacteria, including drug-resistant ones, to enriched soils and groundwater after the natural use of sewage sludge. Of the four applied doses, only the lowest one (10 t/ha) did not cause sanitary contamination of the environment.

2. Material and methods

2.1. Research facilities

The examinations of the migration of drug-resistant microorganisms from sewage sludge to fertilized soil and then to leachate from the soil were conducted as part of a lysimetric experiment under condition of cultivation in a foil tunnel for one year.

Light soil with granulometric composition of loamy soil was used in the experiment. The pH value in the soil used in the lysimetric experiment was 6.4 and, according to the fertilization recommendations [21], it exhibited poorly acid reaction. Concentration of heavy metals in the soils determined according to the standards [21] and presented in Table 1 was below permissible levels recommended for fertilization with sewage sludge [22].

Table 1

Physical and chemical properties of soil and sewage sludge used in the lysimeter experiment.

Parameter	Unit	Soil	Sewage sludge
Organic matter	[% d. m.]	0.8	47.0
pHH20	-	6.4	8.2
Organic carbon	[g∙kg-1 d. m.]	9.65	230
N (total)		0.65	37.12
P (available)	[mg∙kg -1d. m.]	35.12	611.5
K (available)		19.49	262.4
Mg (available)		59.9	885.4
Chromium (Cr)		1.6	19
Zinc (Zn)		3.6	775
Lead (Pb)		7.1	27
Copper (Cu)		1.1	156
Cadmium (Cd)		0.1	2.6
Nickel (Ni)		0.92	120.1
Mercury (Hg)		0.0018	0.52

d. m.—dry mass.

For fertilization purposes in the lysimetric experiment, the authors used the sewage sludge from two wastewater treatment plants situated in the south of Poland. Sewage sludge was formed after biological treatment of sewage using the activated sludge method. Then it was stabilized aerobically. After this process, sewage sludge was thickened mechanically and dewatered by means of a belt press. After dewatering of sewage sludge on belt presses, it was composted naturally in prisms (without lime additives) on plots on the site of the treatment plant for a period of 6 months.

The experiment was conducted in polyethylene lysimeters with capacity of 10 kg dedicated to sludge sampling. The lysimeters were filled with sandy soil and fertilised with different doses of sewage sludge. The objects were fertilized with the doses calculated per pot so that they corresponded to the amount of 10, 20, 30 and 40 tons of fertilizer per hectare. The control objects with non-fertilized soils were also used in the experimental procedure. The soil mixtures fertilized in this way were sown with Dactylis glomerata grass.

The humidity during the lysimetric experiment was maintained at the level of 60% of maximum water capacity by watering with well water. The soil (collected from lysimeters at the depth of 25 cm) and the leachates collected from the research objects were analysed one year after fertilization with sewage sludge. All the tests were carried out with 3 repetitions. The results are represented by mean levels from these repetitions.

Soil leachates can enter surface water and also cause their microbiological contamination.

2.2. Sanitary analysis of sewage sludge, fertilized soils and infiltrating waters

Sanitary examinations were conducted based on the recommendations for the analysed environments [23]. The sanitary analysis of water was carried out in accordance with the current standards used in Poland [24]. The content of mesophilic bacteria (potentially pathogenic—on a nutrient agar), Salmonella bacteria (on SS medium) [25] and Enterobacteriaceae and Enterococcus bacteria [26, 27] was determined in the sewage sludge used for fertilization, soil fertilized with sewage sludge and water leachates. The coliform index of Escherichia coli [28], Clostridium perfringens [29] and Proteus vulgaris [26, 27] was also determined.

2.3. Examinations of drug resistance of bacteria occurring in sewage sludge, soil and infiltrating waters

The collected research materials (sewage sludge, fertilized soil and water leachate from soils fertilized with sewage sludge) were also subjected to microbiological analyses, which were aimed to determine the quantitative and qualitative composition of drug-resistant microorganisms on ENDO, BEA and nutrient agars (with three repetitions). Incubation of microorganisms was performed for 24 hours at 37°C.

Identification of individual groups of isolated microorganisms used the respective selective agars mediums. Agar medium (MPA) was used to determine the total count of mesophilic (potentially pathogenic) microorganisms in the samples.

After bacteria colonies were grown, they were sieved for three times using the reduction method in order to obtain pure strains. Identification of isolated intestinal bacteria was based on biochemical Microgen GN-ID A + B multitests. Enterococci were identified by means of Microgen Strep ID biochemical multitests. As recommended by the manufacturer of the multitests, enterococci were incubated after inoculation for 24 hours at temperature of 37°C. The results of determinations were analysed by means of Microgen MID 60 software.

Drug resistance of intestinal bacteria and enterococci isolated from the environments studies was examined using the diffusion-disc method. The Mueller-Hinton agar recommended in clinical diagnostics was used. In the case of intestinal bacteria, we performed the analysis of drug susceptibility to amikacin, co-amoxiclav, cefazolin, ceftazidime, cefuroxime, ciprofloxacin, ampicillin and gentamicin. In the case of determination of drug resistance in enterococci, tests were performed to examine susceptibility to ampicillin, ciprofloxacin, penicillin, erythromycin, streptomycin, vancomycin, chloramphenicol, tetracycline, linezolid and imipenem. The drugs used are most commonly used to combat these groups of microorganisms [26, 27].

After even spreading of the suspensions of the bacteria isolated on the Mueller-Hinton agar surface (on Petri dishes), the discs soaked with the respective antibiotics with recommended concentrations were applied and the incubation was performed at the temperature of 37°C for one day. The results concerning susceptibility to individual antibacterial compounds were read from the interpretation tables for minimal inhibiting concentrations and the size of growth inhibition zones developed by the European Committee on Antimicrobial Susceptibility Testing [30].

3. Results

Physical, chemical and sanitary characterization of materials used in the study is presented in Tables 1 and 2.

Table 2

Results of sanitary examinations of soil and sewage sludge used for the experiments.

The type of material to be tested	Determination
	Bacteria count			Total bacteria count [CFU/1ml]
	Escherichia coli	Clostridium perfringens	Proteus vulgaris	Mesophilic	From the family Enterobacteriaceae	From the family Enterococcus	Salmonella sp.
Soil	n. d.	n. d.	10−1	3.9·103	n. d.	n. d.	n. d.
Sewage sludge	10−6	10−5	10−5	20.1·109	1.2·107	6.5·101	n. d.

n. d.- not detected, CFU—colony forming unit.

Light sandy soil was used for the examinations Its reaction was 6.4 (Table 1) and, according to fertilizing recommendations [21], it exhibited poorly acid reaction. The content of chromium and mercury in the control soil was within the range of the permissible concentration in soil. Taking into account to the Institute of Soil Science and Plant Cultivation (IUNG) guidelines used to assess the degree of heavy metals contamination of soils with heavy metal, the contents of standardized metals such as zinc, lead, copper, cadmium and nickel in sandy soil used for fertilization in the lysimeter experiment could be determined as a natural quantity (0 degree of soil contamination) [31]. Concentration of heavy metals in the soils was below permissible levels recommended for fertilization with sewage sludge [22].

Table 2 presents the results of sanitary examinations used for lysimeter experiments of soil and sewage sludge.

The analysis of the results of the data presented in Table 2 showed that sandy soil did not contain sanitary contaminants. According to the recommendations [23], it could be classified as clean soil. The sewage sludge used in the experiment did not contain bacteria of the Salmonella sp. genus and could be used for fertilizing purposes.

Tables 3 and 4 present the results of sanitary tests of soil fertilized with sewage sludge and leachates from these soils. The results of quantitative analyses of mesophilic bacteria and Enterobacteriaceae and Enterococcus families obtained one year after fertilization are also presented.

Table 3

Results of sanitary examinations of soil fertilized with different doses of sewage sludge (after completion of the experiment).

Dose of sewage sludge applied for soil fertilization [t/ha]	Determinations in soil fertilized with sewage sludge
	Bacteria titer			Total bacteria count [CFU/1ml]
	Escherichia coli	Clostridium perfringens	Proteus vulgaris	Mesophilic	From the family Enterobacteriaceae	From the family Enterococcus
0	n. d.	n. d.	10−1	2.4·103	n. d.	n. d.
10	10−1	10−2	10−3	3.5·105	5.2·104	2
20	10−1	10−2	10−4	4.5·106	3.7·105	6
30	10−3	10−3	10−4	6.2·106	4.3·105	9
40	10−4	10−3	10−5	5.53·107	7.5·105	12

n. d.—not detected, CFU—colony forming unit.

Table 4

Results of sanitary examinations of water leachates from soil fertilized with different doses of sewage sludge (after completion of the experiment).

Dose of sewage sludge applied for soil fertilization [t/ha]	Determinations in water leachates from soil fertilized with sewage sludge
	Bacteria titer			Total bacteria count [CFU/1ml]
	Escherichia coli	Clostridium perfringens	Proteus vulgaris	Mesophilic	From the family Enterobacteriaceae	From the family Enterococcus
0	n. d.	n. d.	n. d.	2.2·101	n. d.	n. d.
10	10−2	10−1	10−1	1.6·102	1.5·101	n. d.
20	10−2	10−2	10−3	2.7·103	4.2·102	3
30	10−3	10−2	10−3	3.5·103	7.3·103	5
40	10−3	10−3	10−4	1.7·104	4.8·103	8

n. d.—not detected, CFU—colony forming unit.

Analysis of the results presented in Table 3 found that the fertilization with sewage sludge at all doses affected the sanitary condition of the fertilized sandy soil. Only non-fertilised control soil could be classified as sanitary clean. The amount of mesophilic bacteria determined in soils fertilized with 20, 30 and 40 t/ha suggests soil contamination. The total number of bacteria of above 2.5·106 according to the pattern for the assessment of soil sanitary condition indicated its pollution [23]. Similarly, the determination of other sanitary indices, including Escherichia coli indicates low contamination after application of doses of 10 and 20 t/ha and strong contamination of fertilized soils after application of doses 30 and 40 t/ha. Similar observations were found for anaerobes (Clostridium perfringens). There was also an increase in the number of spoilage bacteria in the fertilized soils, which was evidenced by the Proteus vulgaris. The use of sewage sludge also caused a significant increase in the number of intestinal bacteria from the Enterobacteriaceae family.

The results presented in Table 4 prove that microorganisms from fertilizing materials, including sewage sludge, are likely to migrate to soils and further to groundwater. The increase of mesophilic (potentially pathogenic) and intestinal Enterobacteriaceae bacteria was determined in the leachates from sandy soil fertilized with sewage sludge. The lowest contamination was found after the application of the dose of 10 t/ha.

Table 5 presents the results of tests of resistance to selected antibiotics of bacterial species isolated from sewage sludge. Tables 6–8 show the results of drug resistance of bacteria isolated from soils and water leachates (from soil mixtures) fertilized with sewage sludge.

Table 5

Results of antibiograms for individual bacteria from the Enterobacteriaceae family and saprophytic bacteria isolated from sewage sludge.

Bacteria species isolated from sewage sludge	Type of antibiotic used
	Co-amoxiclav (AMC30)	Amikacin (AK30)	Gentamicin (CN10)	Cefazolin (CZ30)	Ciprofloxacin (CIP5)	Ceftazidime (CAZ30)	Ampicillin (AM10)	Cefuroxime (CXM30)
Citrobacter freundii	S	S	S	S	S	S	MS	S
Morganella morganii	R	S	S	R	S	S	R	MS
Klebsiella pneumoniae	S	S	S	S	S	S	MS	S
Klebsiella oxytoca	S	R	S	S	S	S	R	S
Yersinia aldovae	S	S	S	S	S	S	R	S
Yersinia enterocolitica	R	S	R	S	S	R	R	S
Serratia marcescens	S	S	S	R	S	S	S	S
Serratia rubidaea	S	S	S	S	S	S	MS	S
Burkholderia pseudomallei	R	S	S	S	S	S	R	S
Pseudomonas fluorescens *	R	S	S	S	S	S	S	S
Pseudomonas stutzeri *	S	S	S	S	S	S	R	S
Alcaligenes faecalis *	S	S	R	S	S	S	S	R
Photorhabdus luminescens	S	S	S	S	S	S	R	S
Proteus vulgaris	S	S	S	R	S	MS	R	R
Providencia rettgeri	S	S	S	S	S	S	R	S
Providencia stuartii	R	S	S	R	S	MS	R	R
Enterobacter kobei	S	MS	S	S	S	S	R	S
Eschericha coli	S	S	S	S	S	S	S	S
Eschericha coli	R	S	S	S	S	S	R	S
E. coli—inactive L+	R	S	S	S	S	S	R	S
E. coli—inactive L-	S	S	S	S	S	S	R	S
Enteric Group	R	S	S	S	S	S	R	S

Symbols used for susceptibility of bacteria to antibiotics: S—susceptible, MS—medium susceptible, R—resistant.

species that are typically saprophytic in the environment.

Table 6

Results of antibiograms for individual bacteria from the Enterobacteriaceae family and saprophytic bacteria isolated from soil after a year from fertilization with sewage sludge.

Bacteria species isolated from soil	Dose of sewage sludge applied for soil fertilization [t/ha] **	Type of antibiotic used
		Co-amoxiclav (AMC30)	Amikacin (AK30)	Gentamicin (CN10)	Cefazolin (CZ30)	Ciprofloxacin (CIP5)	Ceftazidime (CAZ30)	Ampicillin (AM10)	Cefuroxime (CXM30)
Citrobacter freundii	20,30,40	S	S	S	S	S	S	R	S
Morganella morganii	10,20,30,40	R	S	S	R	S	S	R	MS
Klebsiella pneumoniae	10,20,30,40	S	S	S	S	S	S	MS	S
Klebsiella oxytoca	30,40	S	MS	S	S	S	S	R	S
Yersinia enterocolitica	10,20,30,40	R	S	R	S	S	R	R	S
Serratia marcescens	10,20,30,40	S	S	S	R	S	S	MS	S
Serratia rubidaea	20,30,40	S	S	S	MS	S	S	MS	S
Burkholderia pseudomallei	20,30,40	R	S	S	S	S	S	R	S
Pseudomonas fluorescens *	10,20,30,40	MS	S	S	S	S	S	S	S
Alcaligenes faecalis *	20,30,40	S	S	R	S	S	S	R	R
Eschericha coli	10,20,30,40	S	S	S	S	S	S	S	S
Eschericha coli	30,40	MS	S	S	S	S	S	R	S
E. coli—inactive L+	10,20,30,40	R	S	S	S	S	S	R	S
E. coli—inactive L-	10,20,30,40	S	S	S	S	S	S	R	S
Enteric Group	20,30,40	MS	S	S	S	S	S	R	S

Symbols used for susceptibility of bacteria to antibiotics: S—susceptible, MS—medium susceptible, R—resistant.

species that are typically saprophytic in the environment.

** the type of the dose used for fertilization in which the microorganism was found to be present.

Table 8

Results of antibiograms for bacteria from the Enterococcus genus isolated from the sewage sludge, soil fertilized with sewage sludge and water leachates.

Species of isolated bacteria		Chloramphenicol (C30)	Ciprofloxacin (CIP5)	Ampicillin (AM10)	Erythromycin E15	Penicillin (P10)	Streptomycin (S300)	Linezolid (LNZ30)	Tetracycline (TE30)	Vancomycin (VA30)	Imipenem (IMP10)
Sewage sludge	E. faecalis	S	S	S	R	MS	R	R	S	S	S
	E. faecium	S	S	S	MS	MS	S	S	S	S	S
	E. gallinarum	S	S	S	MS	MS	S	S	S	MS	S
Soil (doses 30 and 40 t/ha) **	E. faecalis	S	S	S	R	MS	R	MS	S	S	S
	E. faecium	S	S	S	MS	MS	S	S	S	S	S
	E. gallinarum	S	S	S	MS	MS	S	S	S	S	S
Water leachates (doses 40 t/ha) **	E. faecalis	S	S	S	R	MS	MS	MS	S	S	S

Symbols used for susceptibility of bacteria to antibiotics: S—susceptible, MS—medium susceptible, R—resistant.

** the type of the dose used for fertilization in which the microorganism was found to be present.

The data in Tables 2 and 5 show that Enterobacteriaceae are the most abundant intestinal bacteria in sewage sludge. A smaller group of bacteria are Enterococcus family. Few species were isolated from this group of organisms: E. faecalis, E. faecium and E. gallinarum (Table 8). All three species were found in sewage sludge and fertilized soil. The species migrating to and determined in soil was E. faecalis. An increase in the number of these microorganisms in the soil with an increase in the fertilization dose was observed. Sanitary parameters determined in soil leachates indicated significant exceeding of parameters in relation to waters used for drinking [32] and bathing purposes. According to the standards, water used e.g. for bathing purposes should not contain more than 100 CFU per 100 ml (recommended value) of Escherichia coli or fecal coliform bacteria (thermotolerant coliforms) and fecal streptococci (enterococci).

After the application of doses of 20, 30 and 40 t/ha of sewage sludge to the soil, the level of water infiltrating the fertilized soil did not meet the above requirements. A worrying phenomenon was the observed resistance of E. faecalis bacteria to erythromycin (Table 8).

The examinations showed that sewage sludge is a significant source of drug-resistant Enterobacteriaceae bacteria in organic soils fertilized with this waste (Tables 5 and 6). It was also shown that these microorganisms may migrate together with soil leachate to groundwater, posing a real threat to the environment (Table 7). The data in Table 7 also show that fertilization with the lowest dose (10 t / ha) of sewage sludges did not cause significant contamination of sandy soil and water leachate with drug-resistant pathogenic bacteria from Enterobacteriaceae family. The use of higher doses of sewage sludges increased of soil and water pollution with these microorganisms.

Table 7

Results of antibiograms for individual bacteria from the Enterobacteriaceae family and saprophytic bacteria isolated in water leachates from soil fertilized with sewage sludge.

Bacteria species isolated from water	Dose of sewage sludge applied for soil fertilization [t/ha] **	Type of antibiotic used
		Co-amoxiclav (AMC30)	Amikacin (AK30)	Gentamicin (CN10)	Cefazolin (CZ30)	Ciprofloxacin (CIP5)	Ceftazidime (CAZ30)	Ampicillin (AM10)	Cefuroxime (CXM30)
Morganella morganii	20,30,40	R	R	S	R	S	S	R	MS
Klebsiella pneumoniae	20,30,40	S	S	S	S	S	S	MS	S
Klebsiella oxytoca	20,30,40	S	S	S	S	S	S	R	S
Yersinia aldovae	30,40	S	S	S	S	S	S	R	S
Yersinia enterocolitica	20,30,40	R	S	R	S	S	R	R	S
Serratia marcescens	20,30,40	S	S	S	R	S	S	S	S
Serratia rubidaea	30.40	S	S	S	S	S	S	MS	S
Pseudomonas fluorescens *	10,20,30,40	R	S	S	S	S	S	S	S
Alcaligenes faecalis *	20,30,40	S	S	R	S	S	S	R	MS
Providencia rettgeri	30,40	S	S	S	S	S	S	MS	S
Eschericha coli	10,20,30,40	S	S	S	S	S	S	S	S
Eschericha coli	20,30,40	MS	S	S	S	S	S	R	S
E. coli—inactive L+	20,30,40	R	S	S	S	S	S	R	S
E. coli—inactive L-	20,30,40	S	S	S	S	S	S	MS	S
Enteric Group	10,20,30,40	R	S	S	S	S	S	R	S

Symbols used for susceptibility of bacteria to antibiotics: S—susceptible, MS—medium susceptible, R—resistant.

species that are typically saprophytic in the environment.

** the type of the dose used for fertilization in which the microorganism was found to be present.

4. Discussion

Analysis of the data presented in Tables 5–8 indicates a significant risk of various diseases in humans and animals in contact with sewage sludge used in the experiment, fertilized soils and soil leachates. The sanitary condition of the sewage sludge depends on the type of technological processes used to treat wastewater. The most frequent processes used to limit the amount of pathogens include methane fermentation of both wastewater and sewage sludge [33, 34]. Further hygienization of this waste can be achieved by e.g. liming or composting [35].

In the case of the bacteria from Enterobacteriaceae and Enterococcus families determined in the examinations of sanitary indices, their presence was demonstrated in the analysed samples of sewage sludge, fertilized soil and water leachates. Intestinal bacteria count (Enterobacteriaceae) was at a worrying high level. Presence of enterococci was also found (Table 8). However, only very small contents were observed for these bacteria, especially in the soil fertilized by sewage sludge.

Furthermore, no excessive migration to groundwater was found with increasing the dose of the sludge (Tables 3 and 4). Therefore, they do not represent a major epidemiological problem in the examined environments according to the recommended standards. Only E. faecalis migrated to soil leachates fertilized with sewage sludge from three bacterial species of the Enterococcus family determined in sewage sludge. Despite a low count of bacteria from this species, their resistance to erythromycin and mean resistance to penicillin, streptomycin and linezoid were determined. Similar results were obtained by Da Silva et al. [36]. In the raw wastewater, these researchers found Entercoccus hirae, Entercoccus faecium and Entercoccus faecalis. A decrease in the count of Entercoccus hirae bacteria and an increase in the count of Entercoccus faecium and Entercoccus faecalis were found in the treated municipal wastewater. Both species were characterized by 40% resistance to erythromycin. Drug-resistant strains of enterococci were not eliminated through wastewater treatment. Da Costa et al. also isolated Enterococcus bacteria in sewage and sewage sludge which showed 24.8% resistance to erythromycin. In 49.4%, they also showed multi-drug resistance [37]. These are bacteria that represent a serious epidemiological threat [38, 39].

In this study, the doses of sewage sludge of 10, 20, 30 and 40 t/ha did not cause contamination by these microorganisms. However, there is a risk that microbiological contamination with drug-resistant forms may already be significant after the application of higher doses.

The group of intestinal bacteria from the Enterobacteriaceae family dominated in the sewage sludge used for fertilization (Table 2). Among them, many isolated species (Table 5) have shown resistance to the antibiotics used. These are the drugs that are most often used to combat these forms of microorganisms. Ampicilin turned out to be the least effective drug for the determined bacteria. Among the isolated species, 68.2% showed resistance to this drug, while 13.6% showed average resistance. Only 18.2% of the intestinal bacteria tested in the sewage sludge were sensitive to this antibiotic. Similarly high ampicilin resistance in Enterobacteriaceae was observed in a study by Mahmud et al. [40].

Analysis of the data of the content of pathogenic drug-resistant bacteria of Enterobacteriaceae family and saprophytes in sewage sludge (Table 5) and comparison with isolated microorganisms from the soil fertilized with them (Table 6) and in infiltrating ground water (Table 7) revealed that the microbial forms tested were actively migrating in the environment. No pathogenic forms were isolated from the control soil. The application of sewage sludge doses above 10t/ha caused the appearance of drug-resistant Escherichia coli (a conditional pathogen) and other intestinal bacteria in the fertilized soil and ground water. It is noteworthy that pathogenic drug-resistant bacteria of the Klebsiella genus were isolated from both sewage sludge and the soils and groundwater fertilized with it. Some Klebsiella species are a natural part of the human intestinal flora and of the oral and nasal cavities. They represent opportunistic pathogens. The Klebsiella pneumoniae bacteria (sometimes called a superbug) can cause several serious illnesses (acute pneumonia often leading to death, urinary tract infection, and even sepsis) in weakened organisms. Many researchers describe it as highly resistant to most antibiotics (some scientists even believe that they are resistant to all antibiotics, as is the case with the New Delhi strain). Another dangerous bacterium isolated from soil and groundwater (after application of sewage sludge doses higher than 20 t/ha) was Klebsiella oxytoca. This bacterium may be responsible for endocolitis and sepsis [20].

Particularly dangerous are Klebsiella oxytoca species, considered to be alert bacteria. They can cause haematosepsis and various infections. Its resistance to the amikacin and ampicillin used in the experiments may be dangerous. These organisms may migrate to soils fertilized with sewage sludge and further to groundwater, which was confirmed in the study. This phenomenon was particularly observed after the application of doses of sewage sludge of 30 and 40 t/ha. A similar phenomenon was observed in the case of the equally dangerous Klebsiella pneumoniae bacteria. Both species very quickly and easily become resistant to most antibiotics. Other experiments performed by the authors [18] also indicated the likelihood of presence and migration of these bacteria from the sewage sludge to soil, especially if higher doses of sewage sludge are applied. An increase in the degree of resistance of isolated bacteria was also observed at that time.

In our research, 36.6% of the isolated bacteria from the Enterobacteriaceae family and saprophytic bacteria isolated from sewage sludge, soil and water were resistant to co-amoxiclav. Ciprofloxacin (100% sensitivity) proved to be the most effective antibiotic against the determined microorganisms.

Among the isolated bacteria from the Enterobacteriaceae family, the species Escherichia coli occurred in large counts. The studies found different groups of these microorganisms that differed in reaction to the antibiotics used. Some were entirely sensitive to all drugs. Others, however, showed resistance mainly to co-amoxiclav and ampicillin. Differences in the reaction were also observed in relation to the presence of unstable forms of L+ and L- among isolated E. coli bacteria. Some authors [41] have demonstrated that L-forms (with cells of this shape), also called wild strains, are resistant to penicillin. The studies also showed resistance to ampicillin.

Some E. coli bacteria can contaminate plant biomass through soil and water. Multi-drug-resistant strains are particularly dangerous [42]. Other authors found the presence of other drug-resistant Acinetobacter bacteria in the biomass of lettuce and fruit [43].

Other dangerous bacteria isolated from the sewage sludge that migrated to soil and groundwater were Serratia marcescens and Serratia rubidaea. S. marcescens bacteria were initially considered non-hazardous saprophytic microorganisms, occurring mainly in aquatic environments. The first documented cases of human infection were found in the area of Great Britain at the beginning of the 20th century. Since then, further reports of urinary tract and endocardial infections, meningitis and sepsis have also been documented. Some researchers demonstrated a substantial intensification of the problem of hospital-acquired conditions caused by S. marcescnes in neonatal and paediatric wards. Depending on the centre studied, S. marcescens bacteria account for between 5 and 16% of nosocomial infections among newborns and infants [44]. The species S. marcescens are microorganisms resistant to numerous groups of antibiotics. Resistance to cefazolin was found in the present study. The migration of these bacteria to soils fertilized with sludge and groundwater seems to be worrying.

Yersinia bacteria were detected in sewage sludge, fertilized soils and infiltrating water. Isolated species of Y. enterocolitica and Y. aldovae can cause many diseases. Y. enterocolitica bacteria cause yersiniosis, systemic infections and haematosepsis, which are dangerous for human health. Isolated in the study, these bacteria turned out to be resistant to many antibiotics including: co-amoxiclav, gentamicin, ceftazidime and ampicillin.

The results of the study showed that the fertilization with sewage sludge at all doses affected the sanitary condition of the fertilized sandy soil. Only non-fertilised control soil could be classified as sanitary clean. The lowest level of contamination was found after application of the dose of sewage sludge of 10 t/ha.

The research indicates a significant problem of the risk of occurrence of various diseases in humans and animals in contact with sewage sludge and with soil and soil leachates fertilized with this sludge. Many drug-resistant bacteria were isolated from the material studied, including Klebsiella oxytoca, which is considered to be an "alert bacterium".

The results show that pathogenic microorganisms, including those drug-resistant, can migrate from sewage sludge to soil and further to groundwater.

Most often isolated drug-resistant strains of intestinal bacteria were less sensitive to older types of antibiotics, including cefazolin, ampicillin and co-amoxiclav.

Resistance of several isolated strains of intestinal bacteria to newer antibiotics, e.g. ceftazidime is worrying. However, the determined bacteria were not resistant to ciprofloxacin.

17 Sep 2021

PONE-D-21-26629Analysis of migration of pathogenic drug-resistant bacteria to soils and groundwater after fertilization with sewage sludgePLOS ONE

Dear Dr. Stańczyk-Mazanek,

Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.Please submit your revised manuscript by Nov 01 2021 11:59PM. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org. When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

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We look forward to receiving your revised manuscript.

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Reginald B. Kogbara, Ph.D.

Academic Editor

PLOS ONE

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When submitting your revision, we need you to address these additional requirements.

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Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Partly

Reviewer #2: Yes

**********

2. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: N/A

Reviewer #2: N/A

**********

3. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: Yes

Reviewer #2: Yes

**********

4. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: Yes

Reviewer #2: Yes

**********

5. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: The article entitled "Analysis of migration of pathogenic drug-resistant bacteria to soils and ground water after fertilization with sewage sludge" aimed to study the effect of using sewage sludge as fertilizer for contamination with drug-resistant bacteria in the environment.

In my opinion, the article needs major revision for publication in PLOS one as the structure and quality of the manuscript is not up to quality. The experiment used a novel approach but fail to design and represent the study in a scientifically coherent manner.

The abstract is not organized in a coherent manner. There is no proper introduction and concluding remark. Author should provide a summary of what is known, the rationale for the study, the method used, the outcome obtained, and conclusions drawn.

The introduction should focus on more how sewage sludge led to migration of pathogenic drug-resistant bacteria, the consequence and why the experiment need to be done

The materials and methods should be written in a way of how experiment is done and condensed by removing description not related to materials and methods.

In table 3 and 4, I am not sure why increased dose of sewage sludge applied for soil fertilization would lead to decreased CFU of bacterial strain such as E. coli.

For Table 5, 6 7 and 8, it does not make any sense to evaluate the antimicrobial resistant pattern of a single strain as it will never represent the while scenario. Author should test more random colony from the same sample to make a logical conclusion. Author should find a way to compare results of table 5, 6 and 7 together.

The results should be quantitatively compared in conclusion with other published data.

Reviewer #2: Analysis of migration of pathogenic drug-resistant bacteria to soils and groundwater after fertilization with sewage sludge

The study entitled “Analysis of migration of pathogenic drug-resistant bacteria to soils and groundwater after fertilization with sewage sludge” deals with one of the most alarming threat connected with the environmental safety and human and animal health. The strong side of this article is the duration devoted to the experiment and the genus identification of the obtained strains with antibiotic-resistance analysis in the further part of research. All the needed data is acquired in the manuscript. The manuscript is well organized and it is carrying the proper scientific soundness. However, in some places, there is no precise description of results, errors in discussion, and vocabulary mistakes throughout the manuscript. Here follows the detailed questions and concerns which could help with correction of the manuscript.

Abstract line 13-23:

The Abstract contained well described aim of the study and used methods, but clearly it is lacking the brief characterization of most important results.

Materials and Methods line 99-100

Authors based sanitary analysis on Polish laboratory exercises book (references number 23), in my opinion this could be not sufficient. I encouraged authors to make references on what detailed regulation they based their methods.

Materials and Methods line 135

I think, it is supposed to be “discs soaked” not “dishes soaked”

Results Table 1. line 168-171

In my opinion the Table 1 structure it can be difficult to read which Unit stands for what Parameter. I encouraged to add some additional horizontal lines.

Results line 223-225

In the sentence “According to the standards, water used e.g. for bathing purposes should not contain more than 100 CFU per 100 ml.”, in my opinion this should be clarified to which group of bacteria it refers.

Results line 231-233

The last sentence of the paragraph is unclear and it could be clarified with references to specific Table/Tables.

Results Table 6. and Table 7. line 240 and 252

In those tables I found the column named “Dose of sewage sludge applied for soil fertilization [t/ha]” with it values very unclear. Is this values represent sewage sludge concentrations in which resistant bacteria occurred ? In my opinion additional description should appear.

Discussion line 317-320

Please verify if defined percentage values are correct.

Discussion line 322

Please specify in which samples or stage of the experiment 36.6% of isolated bacteria were resistant to co-amoxiclav.

Manuscript

Please verify the units correctness with SI Units convention.

**********

6. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

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Reviewer #1: No

Reviewer #2: No

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While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com/. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email PLOS at figures@plos.org. Please note that Supporting Information files do not need this step.

27 Oct 2021

Response to Editors and Reviewers

I would like to thank the Editors and both Reviewers very much for their time devoted to reviewing the manuscript. Thank you also very much for the valuable comments in the reviews.

Journal Requirements:

1. The required names of the submitted files were introduced according to the recommendations of the Editors and Reviewers.

2. The Methods section provides additional details on the experimental materials (soil and sewage sludge) that were used for the study.

3. The source of funding for the research and publication comes from the Ministry's funds for scientific research of the Częstochowa University of Technology and it is necessary to provide the information in Acknowledgments: „The scientific research was funded by the statute subvention of Czestochowa University of Technology, Faculty of Infrastructure and Environment”. Such an entry in the publication is necessary for us to receive a refund of the cost of the publication. It is required at the Częstochowa University of Technology. What should I do then? As recommended, I removed Acknowledgments. I am asking You to make the necessary changes on my behalf.

4. "The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript."

5. Answer as in points 3 and 4

6. The manuscript includes the results of the research along with the dataset underlying the findings presented. The data on drug resistance in the tables are specific and therefore presented in this extended form. Therefore, a separate link to the data is not included. They are available in the manuscript. Based on the data contained in the publication, it is possible to reproduce and verify the entire research process.

7. I logged into Editorial Manager with my ORCID ID. I filled in the details.

Responses to Reviewers

Reviewer 1

Thank you for your time and valuable comments in your review.

Changes to the text are highlighted in yellow.

Recommended revisions have been made to the Abstract, Introduction to the study, and Research Methodology.

Answer regarding Tables 3 and 4: in the tables, the amounts of Escherichia coli, Clostridium perfringens and Proteus vulgaris bacteria are given in the form of so-called bacterial titer. The table incorrectly used the description "Bacteria count". The incorrect description has been corrected.

The microbial titer is the smallest volume of test material in which there is at least one living cell of the indicator microorganism. The titer determination is used to determine the degree of microbial contamination of the test material. The coliform titer test is most commonly performed because it is a bacterium that is found in human and animal feces. The coliformtiter determination is the primary method for assessing whether water or another environment (soil) is contaminated with feces.The coliform titer is different from another indicator called the coliform index. The coliform index provides the number of coliform bacteria in 1 dm³. In determining the index, cultures made on Endo medium agar and colonies identified as coliform are counted.

The number of colony-forming bacteria units in the tables described as "Total bacteria count [CFU/1ml]" was determined for the total count of mesophilic bacteria,the Enterococcus and Enterobacteriaceae family.

An increased dose of sewage sludge applied to the soil fertilization caused an increase in the contamination with Escherichia coli of the fertilised soil and the water infiltrating it.

Answer concerning Tables 5, 6, 7 and 8: the results of drug resistance tests are related to various pathogenic groups of microorganisms from different environments tested. Tables 6 - 8 also show which groups of potentially pathogenic microorganisms were found after application of which doses of fertilizing sewage sludge. Data from Tables 5 -7 are compared in the description of the study results. The Reviewer's valuable comments on the form of presentation will be used in planning the next experiments.

Reviewer 2

Thank you very much for your valuable comments on our manuscript. I would also like to thank the Reviewer for appreciating the really large amount of organizational and research work.

Changes suggested and recommended by the Reviewer have been made to the paper.

Changes to the text are highlighted in green.

Abstract line 13-23

A brief characterization of the main results has been added in the Abstract.

Materials and Methods line 99-100

The methodology for sanitary analysis determination has been complemented

Materials and Methods line 135

Corrected as recommended

Results Table 1. line 168-171

The horizontal lines in Table 1 have been added.

Results line 223-225

This part has been complemented

Results line 231-233

The sentence has been removed

Results Table 6. and Table 7. line 240 and 252

The values presented in these tables represent sewage sludge application rates after which resistant bacteria occurred in the aquatic and soil environments.As suggested by the Reviewer, an additional explanatory description has been added below the table.

Discussion line 317-320

Thank you for finding the mistake. A previous value was incorrectly repeated. A correction has been made to the text.

Discussion line 322

A correction has been made to the text.

Manuscript

Units has been checked.

Submitted filename: Response to Reviewers EN.docx

Click here for additional data file.

22 Nov 2021

PONE-D-21-26629R1Analysis of migration of pathogenic drug-resistant bacteria to soils and groundwater after fertilization with sewage sludgePLOS ONE

Dear Dr. Stańczyk-Mazanek,

Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.Please submit your revised manuscript by Jan 06 2022 11:59PM. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org. When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

Please do address the minor comment of Reviewer 2 as it is required for acceptance.

Please include the following items when submitting your revised manuscript:If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter. Guidelines for resubmitting your figure files are available below the reviewer comments at the end of this letter.

A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). You should upload this letter as a separate file labeled 'Response to Reviewers'.

A marked-up copy of your manuscript that highlights changes made to the original version. You should upload this as a separate file labeled 'Revised Manuscript with Track Changes'.

An unmarked version of your revised paper without tracked changes. You should upload this as a separate file labeled 'Manuscript'.

If applicable, we recommend that you deposit your laboratory protocols in protocols.io to enhance the reproducibility of your results. Protocols.io assigns your protocol its own identifier (DOI) so that it can be cited independently in the future. For instructions see: https://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols. Additionally, PLOS ONE offers an option for publishing peer-reviewed Lab Protocol articles, which describe protocols hosted on protocols.io. Read more information on sharing protocols at https://plos.org/protocols?utm_medium=editorial-email&utm_source=authorletters&utm_campaign=protocols.

We look forward to receiving your revised manuscript.

Kind regards,

Reginald B. Kogbara, Ph.D.

Academic Editor

PLOS ONE

Journal Requirements:

Please review your reference list to ensure that it is complete and correct. If you have cited papers that have been retracted, please include the rationale for doing so in the manuscript text, or remove these references and replace them with relevant current references. Any changes to the reference list should be mentioned in the rebuttal letter that accompanies your revised manuscript. If you need to cite a retracted article, indicate the article’s retracted status in the References list and also include a citation and full reference for the retraction notice.

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Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.

Reviewer #1: All comments have been addressed

Reviewer #2: All comments have been addressed

**********

2. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Yes

Reviewer #2: Yes

**********

3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

Reviewer #2: Yes

**********

4. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: Yes

Reviewer #2: Yes

**********

5. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: Yes

Reviewer #2: (No Response)

**********

6. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: Author could have done better in addressing review question but I believe the author efforts are sufficient enough.

Reviewer #2: I am grateful for answering every comment and making corrections. Unfortunately, one of the comments has been wrongly corrected. After solving this issue, the article in my opinion is ready for publication. Therefore, the issue is described below.

- In the revised manuscript Authors removed the sentence in line 260; “A worrying phenomenon was the observed resistance of E. faecalis bacteria to erythromycin.”.

In the Review, I had in mind the other sentence, which is: “It was found that only a dose of 10 t/ha of sewage sludge did not cause significant contamination of sandy soil and infiltrating water.”. I allow myself to recall my comment for this sentence: “The last sentence of the paragraph is unclear and it could be clarified with references to specific Table/Tables.”.

**********

7. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #1: No

Reviewer #2: No

[NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment files.]

While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com/. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email PLOS at figures@plos.org. Please note that Supporting Information files do not need this step.

23 Nov 2021

Dear Reviewer,

Thank You very much for Your valuable comments on our work.

A correction has been made to the text.

Changes to the text are highlighted in green.

Yours faithfully,

Ewa Stańczyk-Mazanek

Submitted filename: Responses to Reviewer 2.docx

Click here for additional data file.

25 Nov 2021

Analysis of migration of pathogenic drug-resistant bacteria to soils and groundwater after fertilization with sewage sludge

PONE-D-21-26629R2

Dear Dr. Stańczyk-Mazanek,

We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.

Within one week, you’ll receive an e-mail detailing the required amendments. When these have been addressed, you’ll receive a formal acceptance letter and your manuscript will be scheduled for publication.

An invoice for payment will follow shortly after the formal acceptance. To ensure an efficient process, please log into Editorial Manager at http://www.editorialmanager.com/pone/, click the 'Update My Information' link at the top of the page, and double check that your user information is up-to-date. If you have any billing related questions, please contact our Author Billing department directly at authorbilling@plos.org.

If your institution or institutions have a press office, please notify them about your upcoming paper to help maximize its impact. If they’ll be preparing press materials, please inform our press team as soon as possible -- no later than 48 hours after receiving the formal acceptance. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information, please contact onepress@plos.org.

Kind regards,

Reginald B. Kogbara, Ph.D.

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

Reviewers' comments:

6 Dec 2021

PONE-D-21-26629R2

Analysis of migration of pathogenic drug-resistant bacteria to soils and groundwater after fertilization with sewage sludge

Dear Dr. Stańczyk-Mazanek:

I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department.

If your institution or institutions have a press office, please let them know about your upcoming paper now to help maximize its impact. If they'll be preparing press materials, please inform our press team within the next 48 hours. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information please contact onepress@plos.org.

If we can help with anything else, please email us at plosone@plos.org.

Thank you for submitting your work to PLOS ONE and supporting open access.

Kind regards,

PLOS ONE Editorial Office Staff

on behalf of

Dr. Reginald B. Kogbara

Academic Editor

PLOS ONE