Effect of Soil Chemistry on Distribution
of Listeria monocytogenes Across Punjab,
Pakistan
Rabia Tahir1*, Masood Rabbani1, Ali Ahmad1,
Muhammad Yasin Tipu2 and Muhammad Zubair Shabbir1
1Department of Microbiology, University of Veterinary and Animal Sciences
Lahore 54600, Pakistan
2Department of Pathology, University of Veterinary and Animal Sciences
Lahore 54600, Pakistan
*Correspondence
author: Rabia.tahir@uvas.edu.pk
Received 24 June 2020; Accepted 26 September 2020;
Published 10 December 2020
Abstract
A comparative study was conducted in Punjab province,
Pakistan to ascertain any correlation between various physico-chemical
characteristics of soil and presence of Listeria monocytogenes (LM) DNA
in soil. For this purpose, 34 soil samples (n=17
positive for LM and n=17 negative for LM) were collected from nine districts of
Punjab province. Atomic absorption
spectrophotometer was used for assessing the levels of several factors like
phosphorous (P), copper (Cu), chromium (Cr) nickel (Ni), manganese (Mn), cobalt
(Co), lead (Pb), cadmium (Cd), iron (Fe), sodium (Na), potassium (K), calcium
(Ca), magnesium (Mg) and nitrogen (N). Contrarily pH, moisture, electrical
conductivity, organic matter, and texture of soil (silt, sand and clay) were
determined by following standard protocols. Upon statistical analysis, a
significant association was observed only for clay (0.000) and organic matter
(0.001) whereas all other factors did not prove any positive association
(P>0.05) with presence of LM DNA in soil. Hence it is concluded that the
composition of soil does influence the existence of LM DNA in environment. ©
2021 Friends Science Publishers
Key words: Chemical factors; Listeria
monocytogenes; Soil; Metal; Pakistan
Introduction
Listeriosis, also named as silage
disease is an infectious condition caused by soil borne Listeria monocytogenes which is prevalent worldwide. It has wide
distribution range and is isolated from water, soil, fruits, vegetables, milk,
meat and dairy products (Locatelli et al.
2013). Listeric infections usually lead
to abortion in ruminants and humans also. Besides abortion, other clinical
manifestations are depression, loss of appetite, fever, septicemia,
encephalitis and ultimately death. The most frequent manifestation of
listeriosis in ruminants is circling in one direction and is referred as
circling disease (Clark et al. 2004). The infection rate of LM in
ruminants is 10 percent while morbidity ranges up to 30 percent (Peter 2000).
Across the world listeriosis occurs in epidemic form and mostly the nature of infection
is subclinical in animals (OIE 2014). In Indian sub-continent, Malik et al.
(2002) has reported some sporadic cases of listeriosis in both humans and
animals. The estimated prevalence of LM in buffaloes by Shakuntala et al. (2006) is 4.4% in India.
In humans, annual endemic disease rate across the world varies from 2 to 15
cases per million of population (McLaughlin et
al. 2011). In the past few years incidence of listeriosis has
increased in elderly people in European countries (Fierer et al. 2001). Australia has constant incidence range
of listeriosis which is 0.2–0.4 cases/100,000 population from 1991–2000 (Botzler et al. 1974), whereas, USA has
seen remarkable rate of 37% decrease in incidence of listeriosis during 1996–2001
(Locatelli et al. 2013). From
Asian countries very few cases are being reported including 48 cases from
Taiwan during 1996–2008 (Kulesh 2017) and
479 cases from China during 1964–2010 (FAO 2004).
Soil is
considered as main source of transmitting several pathogens due to presence of
microbe rich areas named macropores in it (Bundt et al. 2001). In Pakistan
very little work has been documented relevant to LM and no study has ever been
conducted to ascertain role of soil chemistry
with presence of LM in soil. There is no baseline data related to the effects of land management practices on the abundance of
this bacterium and in which soil types or under which environmental conditions
it is more prevalent. this study was designed to correlate data of the prevalence of L. monocytogenes in the soil
with data of soil chemistry for establishing any association.
Materials and Methods
A total of 34 soil samples from LM positive (n=17 and
negative sites (n=17) were taken (unpublished data). Briefly, 200 g of soil
sample was collected from 3 inches below the ground surface with the help of
electronic weighing balance. All the samples were placed into clean,
pre-labeled zipper bags and were transported at room temperature to Department
of Environmental Sciences, University of Veterinary and Animal Sciences, Lahore
and Department of Plant Sciences, Quaid-i-Azam University, Islamabad.
Soil texture was determined by following the protocol of
Robert and Friedrick (1995). The moisture content was measured by
placing sample (10 g) in hot air oven for 4 days at 72°C (Mclean 1982). Digital
pH meter was used for measuring the pH of soil samples (Committee CSS 1978). Ammonium bicarbonate-diethylenetriaminepenta
acetic acid (DTPA) method was used for measuring concentrations of Mg, Cu, Cr,
Ni, Mn, Co, Pb, Cd, Na, Fe, Ca, N (Fierer et al. 2001) and P (Warncke and Brown 1998). The wavelength for
measuring concentration of various analytes in atomic absorption spectrophotometer
SpectrAA-100 (Varian, Springvale Australia) was 880 nm Total soluble salts were
determined by following protocol of Magistad et al. (1945) Whereas
organic matter content was measured by following Nelson and Sommers (1982)
protocol.
Results
The effect of soil composition in relation to presence or
absence of LM was assessed by compiling data into Microsoft Excel spreadsheet.
As the data violated the normal distribution pattern so a non-parametric test
named Mann-Whitney test (95% confidence interval and 5% level of significance)
by S.P.S.S. version 20.0 (S.P.S.S. Inc., Chicago, IL, USA) was utilized.
Considerable variations among concentrations of various analytes were
recorded in soil samples of both LM positive and negative groups (Table 1). A positive
association was observed in case of clay (0.000) and organic matter (0.001)
with L. monocytogenes. Contrarily
analytes like silt (0.918), sand (0.617), pH (0.570), soluble salts (0.318),
Nitrogen (0.364), Phosphorus (0.535), Nickel (0.278), Cadmium (0.959), Copper (0.502),
Manganese (0.570), Calcium (0.270), Magnesium (0.582), Lead (0.052), Sodium (0.263),
Zinc (0.547) and Potassium (0.654) showed no significant association with LM in
soil (Table 2).
Discussion
Soil is
fortified with bacterial DNA, which is liberated actively from bacteria or
after its death the autolytic changes releases it into the environment (Palmen and
Hellingwerf 1995, 1997). Bacterial DNA prevails in the environment for
different range of time period depending upon physical conditions of soil or
type and presence of nucleases in the soil (DeSalle et al. 1992). The
persistence of bacteria in soil is also determined by the texture of soil.
According to study conducted by Marshall (1975), more clay content
in soil enhances the survival rate of bacteria. Locatelli
et al. (2013) in his study also
supported clay soil for enhanced bacterial survival rate due to more ratio of
organic content in it. One other
possible reason for bacterial stability in clay soil is its
Table 1: Characteristics of various
physio-chemical factors of soil in LM positive and negative groups
Soil analyte |
LM positive soil Mean SD |
LM negative soil Mean SD |
pH |
8.528,0.4203 |
8.569,0.4839 |
Soluble salts% |
2.584, 0.6442 |
2.804,0.7500 |
Organic matter |
5.503,3.819 |
11.484, 5.161 |
Calcium (mg/kg) |
0.3131,0.354 |
0.417,0.339 |
Sodium (mg/kg) |
0.119,0.162 |
0.173, 0.144 |
Potassium (mg/kg) |
0.281, 0.3535 |
0.307, 0.3165 |
Nitrogen (mg/kg) |
0.0687, 0.017 |
0.111, 0.160 |
Phosphorus (mg/kg) |
18.63,2.211 |
18.41, 2.44 |
Magnessium (mg/kg) |
0.292, 0.3535 |
0.307, 0.1165 |
Managnese (mg/kg) |
4.329, 2.065 |
3.836,1.468 |
Zinc (mg/kg) |
0.932, 0.367 |
0.865, 0.344 |
Lead (mg/kg) |
6.503,2.819 |
10.184, 7.261 |
Electrical conductivity |
247.80, 88.33 |
269.85,98.8 |
Nickle (mg/kg) |
0.495, 0.585 |
0.247, 0.332 |
Copper (mg/kg) |
0.051, 0.095 |
0.0591, 0.059 |
Clay (mg/kg) |
4.503,4.009 |
10.484, 4.161 |
Silt (mg/kg) |
0.624, 0.214 |
0.497, 0.312 |
Sand (mg/kg) |
0.262, 0.4535 |
0.207, 0.2165 |
Cadmium (mg/kg) |
0.613, 0.2256 |
0.507, 0.3208 |
Table 2: Characteristics of soil and their
association with Positive LM soil samples
Soil analyte |
Mann Whitney U |
*Significance |
pH |
128.000 |
0.570 |
Soluble salts (%) |
115.500 |
0.318 |
Organic matter (%) |
51.500 |
0.001 |
Calcium (mg/kg) |
112.500 |
0.270 |
Sodium (mg/kg) |
112.500 |
0.263 |
Potassium (mg/kg) |
131.500 |
0.654 |
Nitrogen (mg/kg) |
118.500 |
0.364 |
Phosphorus (mg/kg) |
126.500 |
0.535 |
Magnesium (mg/kg) |
116.500 |
0.582 |
Manganese (mg/kg) |
128.000 |
0.570 |
Zinc (mg/kg) |
127.000 |
0.547 |
Lead (mg/kg) |
88.000 |
0.052 |
Electrical conductivity |
116.500 |
0.335 |
Nickle (mg/kg) |
113.000 |
0.278 |
Copper (mg/kg) |
125.000 |
0.502 |
Clay (mg/kg) |
31.000 |
0.000 |
Silt (mg/kg) |
141.500 |
0.918 |
Sand (mg/kg) |
130.000 |
0.617 |
Cadmium (mg/kg) |
143.000 |
0.959 |
*P < 0.05 positively
associated and P > 0.05 vice versa
significant relationship between Base cation saturation
ratio (BCSR). BCSR represents the cations in soil along with the anions
provided from soil. Clay and organic matter both are negatively charged and fix
the nutrients having positive charge which are mandatory for survival of
bacteria (Dowe et al. 1997).
Moreover many studies revealed that fine textured soils are more favorable to
growth of bacteria because fine textured soils
have more pore spaces which protects them from various protozoans (Botzler et al. 1974; McLaughlin et al.
2011). Rate of DNA adsorption is greatly influenced by soil texture
and according to Lorenz and Wackernagel (1994) 100 folds more DNA adsorption is
observed in case of clay soil as compared to sandy soil.
In the present study, soil elements like P, N, Mg, Mn,
Zn, Cd, Ni, Ca, Na, K and Cu did not show any association with presence of LM
in soil. Heavy metals like copper, zinc, cadmium, arsenic, mercury and nickel are
reported to have deleterious effects on bacteria (Lorenz et al. 1991). The untreated industrial waste,
continuously discharged into the surrounding environment, has significantly
increased the level of heavy metals. These elevated levels of heavy metals have
detrimental effects on DNA by inducing cytotoxic effects on living cells. Hence
the soil contaminated with these metals has higher activity of DNAse enzyme
which accelerates the destruction of DNA (Tsuzuki
et al. 1994; Mukherjee and Das 2002) .Chromium is well known to
cause fragmentation of DNA (Patlolla et al.
2009) whereas higher levels of Mg and Ca interfere with the DNA
adsorption level by soil (Nguyen and Chen 2007)
.Cadmium is also lethal but various bacteria have developed cadmium resistance
mechanisms like enzyme systems which render these metals non-toxic. They
protect themselves by stopping their entry within the bacteria cell by
chelating these metal ions and by adopting efflux mechanism. Besides industrial
effluents seepage, soil is contaminated by heavy metals from the fecal material
of birds and animals and decaying vegetation (Basta
et al. 2005). In many villages, the practice of adding animal manure
in fields as fertilizer also leads to heavy metal contamination of soil (Chaney
and Oliver 1996).
Conclusion
Clay and organic matter are associated with the existence
of L. monocytogenes specific DNA in soil. In future studies, controlled
environment and soil type can be used to ascertain the possible role of each
analyte in more efficient way.
Acknowledgements
This study was funded by the Defense Threat Reduction
Agency, Basic Research Award# HDTRA1-01-1-0080 to the Pennsylvania State
University and University of Veterinary and Animal Sciences Lahore, Pakistan.
Author Contributions
M Rabbani, A Ahmad and MZ Shabbir planned the project
while R Tahir and Y Tipu conducted the research and statistical analysis. All
the authors significantly contributed in writing this manuscript.
References
Basta N, J Ryan, R Chaney (2005). Trace element chemistry in
residual-treated soil. J Environ Qual 34:49–63
Botzler R, A Cowan, T
Wetzler (1974). Survival of Listeria monocytogenes in soil and water. J Wildl Dis 10:204–212
Bundt M, F Widmer, M Pesaro, J Zeyer, P Blaser (2001). Preferential flow
paths: Biological ‘hot spots’ in soils.
Soil Biol Biochem 33:729–738
Chaney RL, DP Oliver (1996). Sources, potential adverse effects and
remediation of agricultural soil contaminants. In: Contaminants and the Soil Environment in the Australasia-Pacific Region,
pp:323–359. Springer, Dordrecht, The Netherlands
RG, JM 2004). Listeria monocytogenes gastroenteritis in sheep.
N Z Vet J 52:46–47
Committee CSS (1978). The Canadian System of Soil
Classification. Research Branch, Canada Department of Agriculture
DeSalle R, J Gatesy, W Wheeler, D Grimaldi (1992). DNA sequences from a
fossil termite in oligo-miocene amber and their phylogenetic. Amer J Phys
Anthropol 87:291–301
Dowe MJ, ED Jackson, JG Mori, CR Bell (1997). Listeria monocytogenes survival in soil
and incidence in agricultural soils. J Food Prot 60:1201–1207
FAO - Food and Agriculture Organisation/World Health Organisation.
(2004). Risk Assessment of Listeria
monocytogenes in Ready-to-Eat Foods: Technical Report, Vol. 4, pp:1–78. FAO, Switzerland
Fierer N, JP Schimel, RG Cates, J Zou (2001). Influence of balsam poplar
tannin fractions on carbon and nitrogen dynamics in Alaskan taiga floodplain
soils. Soil Biol Biochem 33:1827–1839
Kulesh R (2017). Prevalence
of Listeria monocytogenes in Ruminants and Victors at Organized Farm and its
Environment. MAFSU, Nagpur, India
Locatelli A, G Depret, C Jolivet, S Henry, S Dequiedt,
P Piveteau, A Hartmann (2013). Nation-wide study of the occurrence of Listeria monocytogenes in French soils using
culture-based and molecular detection methods. J Microbiol Meth 93:242–250
Lorenz MG, D Gerjets, W Wackernagel (1991). Release of transforming
plasmid and chromosomal DNA from two cultured soil bacteria. Arch Microbiol 156:319–326
Lorenz MG, W Wackernagel (1994). Bacterial gene transfer by natural
genetic transformation in the environment. Microbiol Rev 58:563–602
Magistad O, R Reitemeier, L Wilcox (1945).
Determination of soluble salts in soils. Soil Sci 59:65–76
Malik SV, SB Barbuddhe, SP Chaudhari (2002).
Listeric infections in humans and animals in the Indian subcontinent: a review.
Trop Anim Health Prod 34:359–381
Marshall K (1975). Clay mineralogy in relation to survival of soil
bacteria. Annu Rev Phytopathol 13:357–373
McLaughlin HP, PG Casey, J Cotter, CG Gahan, C Hill
(2011). Factors affecting survival of Listeria
monocytogenes and Listeria innocua
in soil samples. Arch Microbiol 193:775–785
Mclean EO (1982). Soil pH and lime requirement. In: Methods of soil analysis, 2nd
edn, Vol. 9, pp:199–223. Page AL (Ed.). Medison, Wisconsin, USA
Mukherjee S, S Das (2002). Acute cadmium toxicity and male reproduction.
Adv Reprod 6:76–76
Nelson DW, LE Sommers (1982). Total carbon, organic carbon, and organic
matter. In: Methods of soil analysis,
pp: 539–579. Page AL (Ed.). Chemical
and microbiological properties, Agronomy Monographs
Nguyen TH, KL Chen (2007). Role of divalent cations in plasmid DNA
adsorption to natural organic matter-coated silica surface. Environ Sci
Technol 41:5370–5375
OIE (2014). Listeria monocytogenes,. In:
Manual of Diagnostic Tests and Vaccines for Terrestrial
Animals, pp:1–18. Paris, France
Palmen R, KJ Hellingwerf (1997). Uptake and processing of DNA by Acinetobacter calcoaceticus – a review. Gene
192:179–190
Palmen R, KJ Hellingwerf (1995). Acinetobacter calcoaceticus liberates
chromosomal DNA during induction of competence by cell lysis. Curr Microbiol
30:7–10
Patlolla AK, C Barnes, D Hackett, PB Tchounwou (2009). Potassium
dichromate induced cytotoxicity, genotoxicity and oxidative stress in human
liver carcinoma (HepG2) cells. Intl J Environ Res Publ Health 6:643–653
Peter A (2000). Abortions in dairy cows:
New insights and economic impact. Adv Dairy Technol 12:233–244
Robert G, R Frederick (1995). Introductory
soil science laboratory manual, p: 120. Oxford University Press, New York,
USA
I, SVS , SB , DB (2006). Isolation of Listeria
monocytogenes from buffaloes with reproductive disorders and its
confirmation by polymerase chain reaction. Vet Microbiol 117:229–234
Tsuzuki K, M Sugiyama, N Haramaki (1994). DNA single-strand breaks and
cytotoxicity induced by chromate (VI), cadmium (II), and mercury (II) in
hydrogen peroxide-resistant cell lines. Environ Health Perspect 102:341–348
Warncke D, JR Brown (1998). Potassium and other basic cations. Recommended chemical soil test
procedures for the North Central Region. Missouri Agricultural Experimental Station, St. Sb 1001, Columbia, USA