antibiotic resistance among enteric … · prevalence of resistance of certain indicator bacteria...

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ANTIBIOTIC RESISTANCE AMONG ENTERIC BACTERIA AND THEIR

HEALTH IMPLICATION

Vishvas Hare*1, Pankaj Chowdhary

2and Vinay Singh Baghel

3

1,2,3Department of Environmental Microbiology, School for Environmental Sciences Babasaheb Bhimrao

Ambedkar University (A Central University), VidyaVihar, Raebareli Road, Lucknow, 226025, Uttar Pradesh,

India.

*Corresponding Author: Vishvas Hare

Abstract

Today antimicrobial agent resistance is an emerging global concern to both public and veterinary

health. The use of antibacterial drugs for prophylactic or therapeutic purposes in humans and for

veterinary and agricultural purposes has provided selective pressure favoring the survival and

spread of resistant organisms. However, resistant bacteria may transfer their resistance to

previously non-resistant pathogenic bacteria or directly infect humans with bacterialdiseases that

cannot be treated by conventional antimicrobial therapies. The potential for antibiotic exposure

and resistance development in human and animal gastrointestinal tracts, coupled with relatively

great abundance in waters contaminated with human and animal waste, makes the fecal coliform

bacteria a logical fecal groupfor studies of antibiotic resistance and transfer in aquatic

environments. The main bacteria present in human and animal feces discussed which indicators

of fecal pollution should be used in current drinking water microbiological analysis.This review

mainly focoused on antibiotic resistance of environmental isolates is imperative to explore the

antibiotic pressure in the environment. In addition methods to reduce bacteria resistant load in

wastewaters and the amount of antimicrobial agents is originated in most cases of hospitals and

farms that optimization the disinfection procedures and management of wastewater.

Key Words: Antibacterial durgs,Bacterial disease, contaminated water,feces, fecal coliform

bacteria,Resistance, wastes water

I. INTRODUCTION

As it is well known that antimicrobial resistance is a global problem in human and veterinary

medicine. Antibiotic resistance in bacteria provides one of the well-documented examples of an

evolutionary response to selection in natural populations. The selective agent can be easily

identified, changes in phenotype have occurred in a human generation, and the mode of transmission

of resistance determinants can be deduced [1-3].The antimicrobial agents used in animal care are

also significant, not only in increasing the resistance in animalpathogens, but also in bacteria

transmitted from animals to humans.Most studies show that not only the level of resistance of

pathogenic bacteria, but alsoof commensal bacteria increases.It is generally accepted that the main

risk factor for increase in the antibiotic resistance is extensive use of antibiotics. This has lead to the

emergence and dissemination of resistant bacteria and resistance genes in animals and

humans.Dramatically rapid increases in antibiotic resistance have been observed in a host of

phylogenetically diverse taxa and the process has occurred repeatedly within taxa as each is

challenged with new antibiotics [1, 4, 5].Antibiotic selection pressure can lead to a rapid reduction

in the initial fitness costs that were obtain as a pleiotropic effect of resistance. Additional

consequences of the rapid evolution of resistance may depend upon population structure. Bacterial

population structures range from distingguishablely clonal to non-clonal, depending on the relative

roles of horizontal and vertical transmission of geneticinformation within a species [4,6].Antibiotic

International Journal of Applied and Pure Science and Agriculture (IJAPSA) Volume 02, Issue 12, [December- 2016] e-ISSN: 2394-5532, p-ISSN: 2394-823X


resistance determinants can spread via either clonalelaboration or horizontal transfer, the latter

mediated by conjugative plasmids, phage vectors, and natural transformation systems.In contrast, the

spread of resistance determinants via promiscuous horizontal exchange can be expected to both

maintain variation and show an absence of linkage disequilibrium between the selected and neutral

markers. Studies based on samples from a single time point often rely upon variation in resistance

genes and linkage disequilibrium to distinguish vertical from horizontal transmission. The

subsequent intraspecific spread of chromosomal resistance genes was most likely mediated by a

combination of clonal elaboration, as evidenced by linkage disequilibrium between resistance genes

and housekeeping genes, and horizontal gene transfer, deduced from the incongruence of

dendrogramsportayaling patterns of variation within the two types of loci [3, 7].Investigation of the

prevalence of resistance of certain indicator bacteria like E. coli and enterococci in the intestinal

tract of different populations of animals and humans makes it workable to compare the prevalence of

resistance in different populations. Because of the in avoidable high usage of antibiotics in hospitals,

selection and diffusion of resistant clones and resistance genes is high in hospitals. Therefore,

healthy individuals in the community outside hospitals hold not only a reservoir of resistant bacteria

and resistant genes, but are considered to be a suitable population to study the possibility of transfer

of resistant bacteria or resistance genes from animals to humans[1]

II. DETECTION OF MICROORGANISM

The detection and reckoning of microorganism trust on cultural methods. In these methods,

the microorganism isgrown on either a solid (agar) or liquid (broth) medium, which supplies the

nutritional requirements of the organisms or in the case of obligate parasites such as viruses, the

organism is grown in a culture of host cells. Once a microorganism has been grown and isolated as a

pure culture, identification of the organism is based on biochemical, immunological (serological),

and genetic characteristics of the isolate. In many illustrations, particularly for such well studied

organisms as the coliforms, specific compounds are obtain into the primary media, which allow for

selection and differentiation of the organisms of interest. For example, Endo agar is routinely used

for the reckoning of coliforms and otherenteric organisms. This agar has sodium sulfite and basic

fuchsinto inhibit the growth of Gram-positive bacteria. In addition, lactose is present as a primary

substrate for bacterial nutrition. Metabolism of lactose with the formation of acid and gas, a

hallmark characteristic of the coliform group, is detected by a color change due to the reaction

between acetaldehyde (an intermediate product of lactose fermentation) and the sodium

sulfite.Within the past several decades alternative, noncultural methods for the detection of

microorganisms have been developed and are now widely used. These methods are particularly

useful for the detection of microorganism for which no selective media has yet been developed.

These methods include the use of specific staining procedures, usually based on serological

properties of the organism (immunofluorescence), and molecular methods for the detection and

characterization of specific sequences within the DNA or RNA of the organism. Immunofluorescent

detection of microorganisms depends on the ability of antibody molecules to recognize and react

with specific three-dimensional regions (epitopes) on the surface of microbial cells. Antibodies can

be labelled with fluorescent dyes. Specific-microorganisms stained with immunofluorescent dyes

can be enumerated by direct counts under an epifluorescentmicroscope.Detection of nucleic acid

sequences unique to a particular organism involve several distingguishable procedures. Nucleic acid

probes containing nucleotide sequences complementary to a unique sequence of a specific

microorganism can be coupled with a variety of reporter molecules (fluorescent dyes and

radioisotopes). When mixed with a solution of DNA elicit from an environmental sample, the probe

will only bind to those specific target sequences that are complementary to it. Thus, the presence of

specific microbial nucleic acid, and presumably then specific microorganisms, can be determined.

Frequently, the amounts of specific nucleic acids present in environmental samples are too low to be

detected directly by gene probes. In this case, there is a need to increase, or amplify, the specific



sequences to detectable levels.This is accomplished through the use of the polymerase chain reaction

(PCR).

III. INDICATOR BACTERIA

Indicator bacteria belonging to the normal intestinal flora of humans and animals. These

bacteria not only constitute an tremendous reservoir of resistance genes for (potentially) pathogenic

bacteria, but also the level of resistance in the endogenous flora is considered to be a good indicator

for the selection pressure exerted by antibiotic use in that population [8] and for the resistance

problems to be expected in pathogens [9].Indicator organisms that can be transmitted by a

waterborne route, (e.g., thermotolerant coliforms) are used widely as deputy for the detection of

pathogens.[10].Compared two media, mFC and mTEC, for the reckoning of fecal coliforms in tidal

creeks.Counts by mTEC were systematically higher than mFC counts at all salinity ranges. In

addition, a significant number of false positives were associated with using mFC in middle and high

salinity areas.Lifshitz and Joshi found the ColiPlate (CP) kit gave estimates of E. coli that were 20%

higher than standard membrane filtration when the two methods were compared for testing water

samples [11]. The difference increased when samples were impaled with injured cells. They

concluded that the CP kit is a more reliablemethod for use with samples having high levels of

injured or weakened cells. m-ColiBlue24 (m-CB) was compared to m-Endo medium and an

International Organization for Standardization (ISO) standard conform medium, lactose agar with

Tergitol 7, for the analysis of indicator organisms in bottled water.Pruss reviewed studies on

uncontrolled waters, such asseas, lakes, and rivers, to evaluate the health risks caused by poor

microbiological quality of recreational water. Most studies reported a dose-related increase of health

risk in swimmers with an increase in the indicator bacteria count in recreational waters. The

indicator microorganisms that correlated best with health out come were Enterococci/Fecal

streptococci for both marine and freshwater and E. coli for freshwater [10].

(1) Fecal Indicator Bacteria

In 1892, Schardinger proposed that since-Bacterium coli was a characteristic component of

the fecal flora, its presence in water could be taken as an indication of the presence of fecal pollution

and therefore of the potential presence of enteric pathogens [12].Various classification schemes for

coliforms have emerged. The earliest were those of MacConkey in 1909, which recognized 128

different coliform types, while Bergey and Deehan in 1908 identified 256. By the early 1920s,

differentiation of coliforms had come to a series of correlations that suggested that indole

production, gelatin liquefaction, sucrose fermentation and the Voges-Proskauer reaction were among

the more important tests for determining fecal contamination. These developments culminated in the

IMViC (Indole, Methyl Red, Voges–Proskauer and Citrate) tests for the differentiation of so-called

fecal coliforms and intermediates [13].Traditional tests for total and fecal coliforms are carried out

either by the multiple-tube fermentation technique or by filtration through membrane. The multiple-

tube fermentation technique is used for medium or highly contaminated waters, and the filtration

through membrane for low or very low contaminated waters. Filtration through membrane is a very

sensitive technique since can detect one (culturable) cell in 500 or even 1,000 mL of water.

However, both methods take several days to complete and do not detect viable but non-culturable

bacteria [14].These limitations stimulate the discovery of alternative methods, faster and, if possible,

less prone to false negative results such as those caused by the viable but non-culturable bacteria.

Fecal Indicator bacteria are generally three types that found on the basis of different test and

describe below following.

3.1.1 Total Coliforms Total coliforms are Gram-negative, oxidase-negative, non-sporeformingrods, which ferment

lactose with gas production at 35-37 °C, after 48h, in a medium with bile salts and detergents [15].

When the test of coliforms is carried out with environmental waters, several species of the four

Enterobacteriaceae genera Escherichia, Klebsiella, Enterobacter and Citrobacter give positive



results and therefore are coliforms according to this definition. However, the environmental

significance of these four genera is very disparate as discussed in the present text. Therefore, total

coliform counts are not necessarily a measure of fecal pollution and indeed can have no relation with

this cause [16].The detection of β-D-galactosidase activity (at 37 °C) is usually a good marker for

total coliforms in environmental waters, since most of these bacteria display this enzymatic activity

[2, 9]. Most Escherichia, Citrobacter, Enterobacter, Klebsiella andRaoultellastrains have

galactosidase. Hafnia, Serratiaand Yersinia also possess this enzymatic activity. Most Proteus,

Salmonella andEdwardsiella strains do not display β-galactosidase [17, 18]. β-galactosidase cleaves

lactose in glucose and galactose, and can be detected by using colored or fluorescent markers that

change color after enzyme action.In environmental waters, the presence of Aeromonas or Vibrio

cholerae can be a source of false positives in the β-D-galactosidase assay, since these bacteria have

galactosidase, but are not coliforms. Additionally, in particular environments, such as estuaries, β-

galactosidase activity can overestimate total coliform count due to UV-stimulated enzymatic activity

in certain bacteria such as E. coli.

3.1.2 Fecal coliforms

Fecal coliforms (or thermotolerant coliforms) are traditionally defined as coliforms that

ferment lactose at 44.5 °C in a medium with bile salt[15].The range of species detected by the

experimental procedure is much lower than that of total coliforms. With environmental polluted

waters, only E. coli, andKlebsellaoxytocaandKlebsellapneumoniae gave positive results in the test

[19].The detection of β-D-glucuronidase activity (at 44.5 °C) is, generally, a good marker for fecal

coliforms in environmental polluted waters and very specific forE.coli[20].In Gram-negative

bacteria, this enzymatic activity if found in most E. coli strains and in some Salmonella and Shigella

strains [21]. Aeromonas, Citrobacter, Enterobacter, non-coli Escherichia, Hafnia, Klebsiella,

Proteus, Serratia, Vibrio, Yersinia, and most Salmonella strains do not display β-glucuronidase

activity [20].β-D-glucuronidase activity can be detected by using colored or fluorescent markers that change color after enzyme action. The presence of this enzyme in some strains of Bacteroides,

Flavobacterium, Staphylococcus, Streptococcus, in anaerobiccorynebacteria and Clostridium, has

also been reported [22]. β-D-glucuronidase activity in fecal bacteria other thenE. coli (Bacteroides,

Bifidobacteria, Clostridia, Enterococci and Lactobacillus) is very limited [23].

3.1.3 Streptococci and Enterococci

Fecal streptococci also belong to the traditional indicators of fecal pollution. Fecal

streptococci are Gram-positive, catalase-negative, non-sporeformingcocci that grows at 35 °C in a

medium containing bile salts and sodium azide[15] Azide is a strong inhibitor of the respiratory

chain. Since streptococci are one of the very few bacteria that have no respiratory chain, the test is

very specific for this group, and false positives are rarely found [24].Fecal enterococci (E. faecalis,

E. faecium, E. avium and E. gallinarum) are fecal streptococci that grow in the presence of 6.5%

NaCl at 45 °C. Selective media use these particular characteristics in order to separate enterococci

from the other streptococci. Several studies have reported on the microbiological composition of

human and animal (cattle, chicken, deer, dog, fowl, goose, and swine) feces. E. faecalisandE.

faecium were present in human and animal feces. However, whereas human feces almost have only

these two enterococci, in the animals others species co-occur, like E. avium, E. cecorum, E. durans,

E. gallinarumandE. Hirae[25].

IV. ANTIMICROBIAL RESISTANCE

Many retrospective and prospective studies have been performed to study the Egression and

selection of resistance in bacteria by antibiotic usage.Despite large differences in methodology, most

results show that after the introduction of an antibiotic in veterinary practise, the resistance in

pathogenic bacteria and the faecal flora increases, as in human medicine.Some bacteria, most

enterobacteriaceae, staphylococci and Pasteurella spp. become more readily resistant to certain

antibiotics than others like Clostridium sps.andstreptococci which are still fully susceptible to



penicillinG.The literature on resistance against APE is very limited as most of these molecules are

not used for therapy and therefore, susceptibility testing is not performed regularly.Linton et al

found a significant increase in the prevalence of resistance against tylosin and bacitracin in faecal

enterococci of fed these molecules [26]. In this study, virginiamycin usage did not result in an

increase in resistance. Ohmae et al. noticed an increase of resistance against carbadox in faecal E.

coli isolates of pigs after its introduction as APE [27]. All resistant isolates from six farms that fed

carbadox continuously to pigs either as APE or for prevention of swine dysentery carried the same

transferable plasmid consultringcarbadoxresistance.Carbadox is not used in poultry and no

carbadoxresistance was found in E. coli isolates from poultry in the same region. Mills and Kelly

[28] also reported an increase in resistance in E. coli isolates from 37 to 61% after the introduction

of carbadox. Carbadox, however, was not only used as an APE, but also for prevention of swine

dysentery and therapy for salmonellosis. Interest in the selection of resistance by APE increased

after the Egression of vancomycin resistant enterococci (VRE) in human infections. It was soon

recognised that avoparcin was until recently commonly used as APE in most EU-member states,

selects for VRE in the intestinal flora of animals. In countries where avoparcin was used as APE,

VRE was not only found in food animals fed with avoparcin, but also in the faecal flora of healthy

humans and pet animals [29, 30].Also resistance against MLS-antibiotics like erythromycin and

quinupristin-dalfopristinis quite common in enterococci from animals fed with related antibiotics as

APE like tylosin (a macrolide) or virginiamycin[30].According to WHO the resistance to antibiotics

is an ability of bacterial population to survive the effect of inhibitory concentration of antimicrobial

agents.Antimicrobial resistance in bacteria may emerge by several pathways. Some bacterial species

are normally and inherently resistant to certain antibiotics, whereas other are sensitive. Sensitivity

has 3 requirements: a target for reaction, a mechanism for transport into the cell before the antibiotic

action takes place and absence of enzymes that could in-activate or modify the antibiotic. A change

in any of these prerequisites could render an antibiotic-sensitive bacterium resistant to the drug

[31].Water bacteria might be endemic to aquatic environments or exogenous, transiently and

occasionally present in the water as a result of shedding from animal, vegetal,or soil surfacess. The

study of antibiotic resistance in endemic water organisms is important, as it might indicate the extent

of modificationof water ecosystems by human action. Aeromonas strains from Portuguese estuarine

water carry less frequently beta-lactamase genes than Enterobacteriaceae[32]. In water reservoirs a-

half of Aeromonas strains might present multiple antibiotic resistance [33].Resistance profiles of

aquatic pseudomonads depend on the species composition, but also from the site in which they were

isolated, being more antibiotic-resistant along shorelines and in sheltered bays than in the open

water, indicating the influence of nonaquatic organisms or pollutants. Nevertheless, such influences

can be found in the more remote water environments; psychrotrophic bacteria from Antarctic show

various degrees of resistance to industrial antibiotics and metals [34].The connexion of antibiotic-

resistance and resistance to heavy metals is very frequent in the same organism (also in the same

plasmid, transposon, or integron) so that industrial pollution probably selects for antibiotic-

resistance and vice versa [35].Indeed metal contamination represents a long-standing, widespread,

and fractious selection pressure for multiresistant organisms. For the nonaquatic organisms,

obviously the density of antibiotic-resistance organisms and antibiotic-resistance genes in fresh

water varies with the proximity to areas with increased antibiotic expenditure, metal pollution, and

between seasons, being more frequently found in rainy seasons [1]. Very little work has been done

to illuminate the role of bacterial biofilms in water environments and its role in antibiotic resistance.

Phenotypic antibiotic resistance in bacterial biofilms might indeed protect the water environment

from selective events caused by the antibiotic release, which probably are acting more effectively on

planktonic bacteria.

4.1 Mechanism of antibiotic resistance in bacteria

The development of antibiotic resistance is the ability of infectious organisms to adapt

quickly to new environmental conditions. Bacteria are single-celled organisms that, compared with

higher life forms, have small numbers of genes. Therefore, even a single random genetic mutation


@IJAPSA-2016, All rights Reserved 3

can greatly affect their ability to cause disease and because most microbes reproduce by dividing

every few hours, bacteria can evolve rapidly. A mutation that helps a microbe surviving to an

antibiotic exposure will quickly become dominant throughout the microbial population. Microbes

also often evolve resistance genes from each other through horizontal gene transfer mechanism

which might enable them to be a multiple antibiotic resistant strain. It is also noted that the

specificity of the interactions between antibiotics and various protein sequences within a bacterium

resultse in significantly high ratio of mutations in its genome which leads to antibiotic resistance.

There is also a relatively high possibility that a particular mutation in a certain target sequence will

result in antibiotic resistance.Antibiotics generally target a variety of essential bacterial functions.

For instance, the β-lactam antibiotics and vancomycininterrupte cell wall synthesis of pathogens,

whereas macrolides and tetracyclines disrupt the protein synthesis at ribosomal level. Bacteria may

develop their antibiotic properties by a variety of mechanisms. One mechanism ofresistance is by

degrading the antibiotic in a step by step process. This degradation starts when bacterial β-

lactamases hydrolyzes the β-lactam ring thus rendering these antibiotics ineffective. A secondary

resistance mechanism is then triggered when the antibiotic target is altered. As the next step, bacteria

may block the entry of antibiotic to the site of action, resulting in decreasedabsorption, which in turn

results in bacteria with decreased sensitivity to vancomycin due to thicker cell walls. Finally,

bacteria may develop efflux pumps that actively pump antibiotics out of the cell so that they do

notreach their target[1].

4.1.1Horizontal gene transfer of antibiotic resistance in water environments

Unlike eukaryotes, prokaryotes do not reproduce sexually, nor do they undergo meiosis.

Horizontal gene transfer occur in prokaryotes have evolved three different mechanism conjugation,

transformation and transduction for creating recombination. Horizontal gene transfer occurs

primarily between members of same species. Horizontal gene transfer will become an important in

evolution of many species. Antimicrobial resistance in bacteria associated with different ecological niches will be a global concern. Theegression of antimicrobial resistant strains of pathogenic

bacteria will become a great threat to the public health. The detection of rising trends in

antimicrobial resistance of bacterial strains facilitates implementation of effective control measures.

The antibiotic susceptibility testing contributes directly to patient care, and of antimicrobial drug

resistance. However, in our region, the study of antibiotic resistance of bacteria from environment

like soil, water or from fish is bare. Therefore, study pertaining to antibiotic resistance of

environmental isolates is imperative to explore the antibiotic pressure in the environment. Methods

to reduce bacteria resistant load in waste waters and the amount of antimicrobial agents is originated

in most cases of hospitals and farms that optimization the disinfection procedures and management

of waste water.

Estuarine water-borne Aeromonas strains carry almost as frequently as Enterobacteriaceae

class 1 integron platforms carrying antibiotic-resistance genes [32].Exclusively environmentally

based organisms, as Delftia, also harbor class 3 integrons.The continuity of such genetic structures

cannot probably be explained entirely by antibiotic selection, suggesting that activities resulting in

antibiotic resistance might have other physiological roles or that they are placed in multifunctional

plasmids. The most frequent gene cassette found involves aminoglycoside- resistance genes, rarely

under positive selection in our days, and there is a distrust that some other resistance genes, as

integronsul genes, might provide benefits for the bacteria, unrelated with resistance. However,some

of these mobile gene cassettes in Aeromonas might involve important mechanisms of resistance, as

Qnr, involved in fluoroquinolone resistance, which might behorizontally propagated by IncU-type

plasmids [36].Certainly the dense bacterial populations in sewage treatment plants favor genetic

exchange among bacterial populations and communities, integrons predating transposons and

plasmid diffusion. Multiresistance plasmids of broad host-range are systematically recovered in

sewage [37]. Interestingly, antibiotic-resistance genes from muck influence the lagoons and

groundwater gene pool, but this pool also contains antibiotic-resistance genes from endemic

bacteria. Aeromonas from aquaculture water systems (fish, eel farming) are particularly resistant to



antibiotics [38] (Penders and Stobbering, found that frequently contain plasmids and integrons with

multiple genes for antibiotic resistance [39],Jacobs and Chenia and the connexion with heavy-metal

resistance is not uncommon [35]. Water originated in transgenic plant fields may constitute a matter

of concern, but no significant differences have been found in bacterial antibiotic-resistance levels

between transgenic and nontransgenic corn fields [40].

V. IMPACT OF ANTIBIOTIC RESISTANCE ON HUMAN HEALTH

Bacteria and other microorganisms that often cause infections are known to be remarkably

resilient and have the ability to develop ways for surviving drugs that are meant to kill or weaken

them. Recent scientific evidence suggests that during the last decade, antibiotic resistance by various

mechanisms has increased worldwide in bacterial pathogens leading to treatment failures in human

and animal infections. However, the resistance against different types of biocides (including

disinfectants, antiseptics, preservatives, sterilants) has been studied and characterized

[41,42].Biocides and antibiotics may share some common behaviour and properties in their

respective activity and in the resistance mechanisms developed by bacteria [43, 44].Although

antibiotic usage has clearly benefited the animal industry and helped providing affordable animal

protein to the growing human population, the use of antibiotics in food production has also

contributed to the Egression and spread of antibiotic multiple resistances (AMR). Along with

antibiotics used for human medicine, the use of antibiotics for animal treatment, prophylaxis and

growth promotion exerts an immeasurable amount of selective pressure toward the egression and

multiplication of resistant bacterial strains. Animals can serve as mediators, reservoirs and

disseminators of resistant bacterial strains and/or AMR genes. Consequently, imprudent use of

antimicrobials in animals may eventually result in increased human morbidity, increased human

mortality, reduced efficacy of related antibiotics used for human medicine, increased healthcare

costs, increased potential for carriage and diffusion of pathogens within human populations and

facilitated Egressionof resistant human pathogens. The patients infected with

pansusceptibleSalmonellatyphimurium are 2.3 times more likely to die within 2 years after infection

than persons in the general Danish population, and that patient infected with strains resistant to

amplicillin, chloramphenicol, streptomycin, suldonamide and tetracycline are 4.8 times more likely

to die within 2 years. Furthermore, they have established that quinolone resistance in this organism

is associated with a mortality rate 10.3 times higher than the general population. It has been well

documented that antimicrobial resistance due to a particular antibiotic used in food animals may

result in reduced efficacy of most or all members of that same antibiotic class, some of which may

be extremely important for human medicine. The current pharmaceutical era faces multi resistant

infectious disease organisms that are difficult and, sometimes, impossible to treat successfully.

When there is an increase in numbers of bacteria that are resistant to antibiotics, it will be more

difficult and more expensive to treat human bacterial infections (Fig 1)[32].

Figure 1.The Human Health Impact of Antimicrobial Resistance in Animal Populations (Adapted from Jalal et al 2012)



VI. EMERGING WATERBORNE BACTERIAL PATHOGENS

The rising pathogenic bacteria of concern outlined here have the potential to be spread

through drinking water, but they do not correlate with the presence of E. coli or with other

commonly used drinking water quality indicators, such as coliform bacteria. In most cases, there are

no satisfactory microbiological indicators of their presence. More studies are needed in order to

understand the real significance and dimension of the diseases caused by water contaminated with

these bacteria, and the ecology of these pathogens [45].

6.1 Escherichia

Escherichia, a member of Enterobacteriaceae, are oxidase-negative catalase-positive straight

rods that ferment lactose. Cells are positive in the Methyl-Red test, but negative in the Voges-

Proskauer assay. Cells do not use citrate, do not produce H2S or lipase, and do not hydrolyze urea

[46]. E. coli is a natural and essential part of the bacterial flora in the gut of humans and animals.

Most E. coli strains are nonpathogenic and reside harmlessly in the colon. However, certain

serotypes do play a role in intestinal and extra-intestinal diseases, such as urinary tract infections

[47].

6.1.1 Pathogenic Escherichia coli Strains

E. coli strains isolated from intestinal diseases have been grouped into at least six different

main groups, based on epidemiological evidence, phenotypic traits, clinical features of the disease

and specific virulence factors. From these, enterotoxigenic (ETEC, namely O148),

enterohemorrhagic (EHEC, namely O157) and enteroinvasive serotypes (EIEC, namely O124) are of

outstanding importance and can be transmitted through contaminated water [47, 48].

6.1.1a EnterotoxigenicE. coli (ETEC) Strains

EnterotoxigenicE. coli (ETEC) serotypes can cause infantile gastroenteritis. The number of

reports of their occurrence in developed countries is comparatively small, but it is an extremely

important cause of diarrhea in the developing world, where there is no tolerable clean water and

poor sanitation. Disease caused by ETEC follows ingestion of contaminated food or water and is

characterized by riotous watery diarrhea lasting for several days that often leads to dehydration and

malnutrition in young children. ETEC also are the most common cause of -travelersdiarrhea that

affects individuals from industrialized countries travelling to developing regions of the World [48,

49].

6.1.1b EnterohemorrhagicE. coli (EHEC) Strains

These organisms produce a toxin known as verocytotoxin which is similar to the toxin

produced by Shigella. Infection with this organism is associated with haemorrhagic colitis. In a

small proportion of the cases, particularly in children, the infection can progress to haemolytic

uraemic syndrome, a life threatening disease. E. coli serotype O157:H7 causes abdominal pain,

bloody diarrhea, and hemolytic uremic syndrome. Although E. coli O157:H7 is not usually a

concern in treated drinking water, outbreaks involving expenditure of drinking water contaminated

with human sewage or cattle feces have been documented. An increasing number of outbreaks are

associated with the expenditure of fruits and vegetables (sprouts, lettuce, coleslaw, salad)

contaminated with feces from domestic or wild animals at some stage during cultivation or handling.

EHEC has also been isolated from bodies of water (ponds, streams), wells and water troughs, and

has been found to survive for months in muck and water-trough sediments [45,49].Person-to-person

contact is an important mode of transmission through the oral-fecal route.

6.1.1c EnteroinvasiveE. coli (EIEC) Strains

EnteroinvasiveE. coli (EIEC) are capable of invading and multiplying in the intestinal

epithelial cells of the distal large bowel in humans. The illness is characterized by abdominal

cramps, diarrhea, vomiting, fever, chills, a generalized malaise, and the appearance of blood and

mucus in the stools of infected individuals. [47, 48]. An investigation in Croatia showed that E. coli



O124 could frequently be isolated from cases of gastroenteritis, enterocolitis, and dysentery. The

dysentery was more common among the older age groups, while the two other types of disease

occurred equally in all age groups.Any food contaminated with human feces from an ill individual,

either directly or via contaminated water, could cause disease in others. Outbreaks have been

associated with hamburger meat and unpasteurized milk.

6.2Salmonella.

Salmonella was discovered between the illness and expenditure of water from an aqueduct

that flowed near thecamp. The risk of suffering from the illness rose with the amount of water

consumed. Chemical and bacteriological analyses of the aqueduct water indicated the presence of

fecal contamination. An outbreak of gasteroenteritis due to S. ohio whose origin was the expenditure

of water from a drinking fountain was described for the first time by [50]. This fountain had no

chlorination system. S. ohio was isolated from the water and from 2 of the 13 stool specimens

analysed. A molecular epidemiology study of Salmonella serotype Enteritidis was carried out by

ribotyping and randomly amplified polymorphic DNA (RAPD) typing of 38 food and 25 water

strains, which were epidemiologically unrelated and collected in Spain from 1985 to 1996 [51] Their

results supported the fact that organisms representing at least 40 genomic groups are currently

circulating in Spain but that only the organisms of five groups preponderant and these fall into a

single subcluster or lineage. Organisms of four infrequentgroups were only collected from sewage or

environmental waters.Typhoid fever, a severe systemic illness transmitted through food or water, is

caused by the bacterium Salmonella serotype typhi. Luby et al. [52] evaluated risk factors for

developing typhoid fever in a setting where the disease is endemic (Karachi, Pakistan). Eating ice

cream, eating food from a roadside cabin during the summer months, taking antimicrobials in the 2

weeks preceding the onset of symptoms, and drinking water at the worksite were all independently

associated with typhoid fever.

6.3 Shigella. Children under 5 years of age infected with either Shigella dysenteriae type I or

Shigellaflexneri attending a diarrhea treatment center from 1993 to 1995 in Dhaka, Bangladesh. Use

of antibiotics at home, use of water from tube wells or pipe-water for drinking, and lack of sanitary

facilities were the behavioral and environmental factors strongly associated with S. dysenteriae type

I infection. Tshimanga et al[53] investigated a July 1994 outbreak of Shigella dysenteriae type I at a

textile factory in Bulawayo, Zimbabwe. Thirty seven of 58 workers who drank borehole water were

ill compared to 1 of the 17 who did not. Water samples from the two boreholes yielded numerous

fecal coliforms.

6.4 Vibrio. Cholera, caused by certain strains of Vibrio cholerae, is the first disease for which a

waterborne route of transmission was shown. Paneth et al[54] reviewed the history of John Snow's

1854 investigation proving a waterborne route of transmission for cholera. Epidemic cholera is

caused by toxigenic strains of V. cholerae, of which strains 01 and 0139 are associated most often,

but not exclusively, with epidemic outbreaks. Faruque et al[55] reviewed the epidemiology,

genetics, and ecology of toxigenic V.cholerae. They emphasized the close connexion among V.

cholera, surface water, and the population interacting with the water.They also noted that molecular

epidemiological studies have disclosed significant clonal diversity among toxigenic strains and

continual Egression of new epidemic clones.

6.5 Campylobacter.

Campylobacter spp.are now recognized as a major cause of gastroenteritis associated with

the ingestion of contaminated food and water. Furtado et al[56] reported that Campylobacter was

associated with the majority of waterborne disease outbreaks from private water supplies. Hazeleger

et al.[57] examined the physiological activities of Campylobacter at several environmental

temperatures. Cellular activity, and hencecontinuity, could be measured at temperatures as low as



40C. In addition, the organism was capable of chemotaxis and aerotaxis at all temperatures and thus

may be able to move to more favourable environments. Both osmolality and temperature were found

to be significant factors in the continuity of Campylobacter spp.[58].None of the Campylobacter

examined (C.jejuni, C. lari, and E. coli) grew in media with an osmolality of 130 mosmol and a

temperature below 42°C. In media with low osmolalities, the number of viable cells declined rapidly

at any of the temperatures examined.

6.6 Acrobacter Arcobacter spp. isolated from the treatment plants showed the same serotypes as described

for human isolates.Therefore, the spread of Arcobacter via the drinking water pathmust be

suspected.Arcobacter enrichment medium (AM), newly developed by Oxoid, was compared with

twoCampylobacter enrichment media (Preston broth [Oxoid] and LabM broth) and with

Arcobacterbasal medium (ABM) as a control by [59] Atabay and Corry.Twenty strains of

ArcobacterandCampylobacter spp. were tested for growth, with target inocula of less than 4

CFU/mL of medium. None of the Campylobacter spp. grew in the complete AM, and only one grew

(very poorly) in the ABM. However, AM supported good growth of all three species of Arcobacter

(A. butzleri, A. skirrowii, and A. cryaerophilus), which have been associated with human and animal

disease.

6.7 Helicobacter pylori

Helicobacter pylori has been cited as a major etiologic agent for gastritis and has been

implicated in the pathogenesis of peptic and duodenal ulcer disease and gastric carcinoma. H. pylori

has not been isolated from environmental sources, including water [60].On the contrary, molecular

methods have been successful in detecting this pathogen. Fluorescence in situ hybridization has been

successfully used to detect this pathogen in drinking water distribution systems and other water

bodies. Polymerase chain reaction has also been used to detect the presence of H. pylori DNA in

drinking water, especially associated with biofilms [61].In drinking-water biofilms, H.pyloricells

rapidly lose culturability, entering a viable but non-culturablestate.How the organism is transmitted

is still not fully understood. However, the fact that it has been recovered from saliva, dental plaques,

the stomach, and fecal samples strongly indicates oral-oral or fecal-oral transmission. Water and

food appear to be of lesser direct importance, but they can still play a significant role in situations

with improper sanitation and hygiene [45]. The survival of Helicobacter pylori in artificially

contaminated milk and tap water was investigated by Fan et al. [62]. Helicobacter pyloricould

survive for up to 10 days in milk at 40C storage but only 4 days in tap water with a steady decrease

of colony-forming units. However, electron microscopy clearly showed that the

nonculturablecoccoid form was present in tap water that had been kept at 4°C for 7 days.

6.8 Mycobacterium avium complex.

Mycobacterium avium complex (MAC) organisms have been isolated from water and soil. It

is now generallyaccepted that environmental sources, especially natural waters, arethe reservoirs for

most human infections caused by MAC.A typical mycobacteria are responsible for a variety of

diseases, particularly in immune compromised individuals. [63] Lin et al. found that Mycobacterium

avium was signify cantly more resistant to disinfection with copper-silver ions

thanwasLegionellapneumophila. Water, both in the city water supplyand hospital environment, was

found to be the major source oftransmission of Mycobacterium xenopi, an opportunistic

pathogenthat causes pulmonary infections [64].Steinert et al. (1998b) [65] compared that the growth

of Mycobacteriumavium in coculture with the free-living amoeba Acanthamoebapolyphaga with the

growth of M. avium when it was separated fromamoebae. Although viable mycobacteria were

observed within amoebal vacuoles, there was no significant difference between bacterialgrowth in

coculture and bacterial growth separately.Mac organisms has ability to survive and grow under

varied conditions. Mac organisms can proliferate in water at temperatures up to 51 °C and can grow

in natural waters over a wide pH range [45].These mycobacteria are highly resistant to chlorine and

the other chemical disinfectants used for the treatment of drinking-water. Standard drinking-water



treatments will not eliminate Mac organisms but, if operating satisfactorily, will significantly reduce

the numbers that may be present in the source water to a level that represents a negligible risk to the

general population. The entryway of these mycobacteria in distribution systems is through leaks.

Growth of Mac organisms in biofilms is probably important for their continuous presence in

distribution systems. The symptoms encountered with Mac infections result from colonization of

either the respiratory or the gastrointestinal tract, with possible diffusion to other locations in the

body. Exposure to Mac organisms may occur through the expenditure of contaminated food, the

inhalation of air with contaminated soil particles, or contact with or ingestion, aspiration, or

aerosolization of potable water containing the organisms [45].With respect to water supplies,

infection with M. avium and M.intracellulare has been well documented. Unlike gastrointestinal

pathogens, where E. coli can be used to indicate their potential presence, no suitable indicators have

been identified to signal increasing concentrations of Mac organisms in water systems [45].

VII. FECAL BACTERIA IN THEIR HOSTS AND IN THE ENVIRONMENT

7.1 Bacteroides

Bacteroides are Gram-negative, non-sporeforming, anaerobic pleiomorphic rods.

Bacteroides are the most abundant bacteria in human feces. In animal feces, on the contrary,

Bacteroides are present at low numbers. Although anaerobic, Bacteroides are among the most

tolerant to oxygen of all anaerobic human gastrointestinal species. B. thetaiotaomicron is one of the

most abundant species in the lower regions of the human gastrointestinal tract. Bacteroides have a

high pathogenic potential and account for approximately two-thirds of all anaerobes isolated from

clinical specimens. The most frequently isolated species has been B. fragilis. The survival of

Bacteroides in environmental waters is usually much lower than the survival of coliforms [66].

7.2 Eubacterium

Eubacterium are anaerobic non-sporeforming Gram-positive rods. Some species have been transferred to other genera-Actinobaculum, Atopobium, Collinsella, Dorea, Eggerthella,

Mogibacterium, Pseudoramibacter and Slackia. Cells are not very aerotolerant. Species isolated

from the human gastrointestinal tract include: E. barkeri, E. biforme, E. contortum, E. cylindrioides,

E. hadrum, E. limosum, E. moniliforme, E. rectal and E. ventricosum.

7.3 Bifidobacterium

Bifidobacteria are Gram-positive, non-sporeforming, pleiomorphicrods.The optimum growth

temperature is 35-39 °C. The genus Bifidobacterium contains ca. 25 species, most of which have

been detected in the human gastrointestinal tract.Bifidobacteria are present in high numbers in the

feces of humans and some animals. Several Bifidobacterium species are specific either for humans

or for animals.Bifidobacteria have been found in sewage and polluted environmental waters, but

appears to be absent from unpolluted or pristine environments such as springs and unpolluted soil.

This results from the facts that upon introduction into the environment bifidobacteriadecrease

appreciably in numbers probably due to their stringent growth requirements.Bifidobacteria grow

poorly below 30 °C and have stringent nutrient requirements. Reports on the survival of

bifidobacteria in environmental waters indicate that their survival is lower than that of coliforms

(Biavati, Mattarelli, 2003 and Wilson UK, 2005).The presence of bifidobacteria in the environment

is therefore considered an indicator of fecal contamination. Since some species are specific for

humans and animals, the identification of Bifidobacterium species present in the polluted water

could, in principle, provide information on the origin of fecal pollution.Bifidobacteria are the less

studied of all fecal bacteria, due to the technical difficulties in their isolation and cultivation. Other

Gram-positive bacteria, such as Streptococcus and Lactobacillus, which may occur in higher

numbers than bifidobacteria, can inhibit their growth. Although selective media has been designed

for the isolation of bifidobacteria from environmental waters, the outcome is still unsatisfactory,

with appreciable numbers of false positives and low recovery percentages (Biavati, Mattarelli, 2003

and Wilson UK, 2005).



7.4 Clostridia

The genus Clostridium is one of the largest genera of the prokaryotes containing 168 validly

published species.Clostridiaare Gram-positive rods, forming endospores. The genus Clostridium

includes psychrophilic, mesophilic, and thermophilic species. Most of the clostridial species are

motile with peritrichous flagellation. Cells are catalase-negative and do not carry out a dissimilatory

sulphate reduction. Clostridia usually produce mixtures of organic acids and alcohols from

carbohydrates and proteins. Many species are saccharolytic and proteolytic. Some species fix

atmospheric dinitrogen. The major role of these organisms in nature is in the degradation of organic

material to acids, alcohols, CO2, H2, and minerals. Frequently, a butyric acid smell is associated

with the proliferation of clostridia. The ability to form spores that resist dryness, heat, and aerobic

conditions makes the clostridia ubiquitous (Wilson UK, 2005, and Hippeet al, 2003).Most species

are obligate anaerobic, although tolerance to oxygen occurs. Oxygen sensitivity restricts the habitat

of the clostridia to anaerobic areas or areas with low oxygen tensions. Growing and dividing

clostridia will, therefore, not be found in air saturated surface layers of lakes and rivers or on the

surface of organic material and soil. Clostridialspores, however, are present with high probability in

these environments, and will germinate when oxygen is exhausted and when appropriate nutrients

are present (Wilson UK, 2005 andHippeet al,2003).C. perfringens ferment lactose, sucrose and

inositol with the production of gas, produce a stormy clot fermentation with milk, reduce nitrate,

hydrolyzegelatin and produce lecithinase and acid phosphatase. The species is divided into five

types, A to E, on the basis of production of major lethal toxins (Rainey et al,2009 and

Smith,2003).C. perfringens appears to be a universal component of the human and animal intestine,

since has been isolated from the intestinal contents of every animal that has been studied. Humans

carry C. perfringens as part of the normal endogenous flora.

7.5 Lactobacillus

Lactobacilli are non-sporeforming Gram-positive long rods. There are more than thirty species in the genus. Most are microaerophillic, although some are obligate anaerobes. Cells are

catalase-negative and obtain their energy by the fermentation of sugars, producing a variety of acids,

alcohol and carbon dioxide. Lactobacilli have complex nutritional requirements and in agarized

media may need the supplementation with aminoacids, peptides, fatty-acid esters, salts, nucleic acid

derivatives and vitamins. Lactobacilli very rarely cause infections in humans (Wilson UK,2005).

7.6 Enterococci

Enterococci are Gram-positive, non-sporeforming, catalase-negative ovoid cells. Cells occur

singly, in pairs or short chains. Optimal growth for most species is 35–37 °C. Some will grow at 42–

45 °C and at 10 °C. Growth requires complex nutrients but is usually abundant on commonly used

bacteriological media. The enterococci are facultative anaerobic but prefer anaerobic conditions

(Wilson UK,2005 and Švec, P.; Devriese, L.A,2009).The genus was separated from Streptococcus in

the 1980s. Enterococci form relatively distingguishable groups. Members of such groups exhibit

similar phenotypic characteristics and species delimitation can be difficult.Enterococci are naturally

present in many kinds of foods, especially those of animal origin such as milk and milk products,

meat and fermented sausages. Enterococci are usually considered secondary contaminants of food,

although they often play a positive role in ripening and aroma development of some types of cheeses

[66, 67] (Wilson UK,2005 and Švec, P.; Devriese, L.A,2009). Although soil is not a natural habitat

for enterococci, cells can be found in this habitat due to the transport by rain.

7.7 Citrobacter

Citrobacter, a member of Enterobacteriaceae, are motile straight rods. Cells are oxidase-

negative, catalase-positive and positive in the Methyl-Red test. Cells use citrate, are negative in the

Voges-Proskauer test and do not decarboxylate lysine [34] (Bergey’s Manual,1994). Citrobacter

species can be isolated from different clinical sites. In particular, C. freundii is intestinal inhabitants

of humans that may sometimes haveevolvedthe ability to produce an enterotoxin and thus become



an intestinal pathogen. Citrobacter is reported to occur in environments such as water, sewage, soil

and food [68] (Frederiksen et al, 2003).

7.8 Klebsiella and Raoultella

Klebsiella and Raoultellaare Enterobacteriaceae, oxidase-negative catalase-positive non-

motile straight rods, surrounded by a capsule. Cells decarboxylate lysine, but are ornithine and

arginine dihydrolase negative. Cells grow on KCN, do not produce H2S and ferment most

carbohydrates [34] (Bergey’s Manual,1994). Klebsiellae are ubitiquous in the environment. They

have been found in a variety of environmental situations, such as soil, vegetation, or water, and they

influence many biochemical and geochemical processes. They have been recovered from aquatic

environments receiving industrial wastewaters,plant products, fresh vegetables, food with a high

content of sugars and acids, frozen orange juice concentrate, sugarcane wastes, living trees, and

plants and plant byproducts. They are commonly associated with wood, sawdust, and waters

receiving industrial effluents from pulp and paper mills and textile finishing plants. Klebsiella have

been isolated from the root surfaces of various plants. K. pneumoniae, K. oxytoca, and R. planticola

are all capable of fixing dinitrogen[69] (Grimont et al, 2005).

7.9 Enterobacter

Enterobacter a member of Enterobacteriaceae, are motile straight rods. Cells are positive in

the Voges-Proskauer test VP and in Simmons citrate agar. Cells do not decarboxylate lysine, but are

ornithine positive. Malonate is usually utilized and gelatin is slowly liquefied. Cells do not produce

H2S, deoxyribonuclease and lipase [34] (Bergey’s Manual,1994).Before the widespread use of

antibiotics, Enterobacter species were rarely found as pathogens, but these organisms are now

increasingly encountered, causing nosocomial infections such as urinary tract infections and

bacteremia. Enterobacter species were the second most common gram-negative organism, behind

Pseudomonas aeruginosa. Both bacteria were reported to each represent 4.7% of bloodstream

infections in intensive care units. Enterobacter species represented 3.1% of bloodstream infections in

non-intensive care units.They found Enterobacter species to be the eighth most common cause of

healthcare-associated infections (5% of all infections) and the fourth most common gram-negative

cause of these infections [70] (Hidron et al, 2008).An Enterobacter cloacae subsp. cloaca (E.

cloacae) occurs in the intestinal tracts of humans and animals, in hospital environments, the skin, in

water, sewage, soil, meat. Nitrogen-fixing strains have been isolated from the roots of rice pdlants.

E. amnigenus has been mostly isolated from water, but some strains were isolated from clinical

specimens from the respiratory tract, wounds and feces. E. asburiae strains were isolated from

clinical specimens, mostly urine, respiratory tract, feces, wounds, and blood [69] (Grimont et

al,2003).

VIII. CONCLUSION

1. It was concluded that safe drinking water for all is one of the major challenges of the 21st

century and that microbiological control of drinking water should be the norm everywhere.

2. In this review a general characterization of the most important enteric bacteria transmitted

through water is presented, focusing on the biology and ecology of the causal agents and on the

diseases characteristics.

3. Currently, it is thought that the input of antibiotics in general as well as from hospitals seems to

be of minor importance, at least in terms of resistance. Up to now, antibiotics have not been

detected in drinking water.

4. There is insufficient information available to reach a final conclusion on the significance and

impact of the presence of resistant bacteria in the environment, which would allow the

assessment of the potential risks related for instance, to human health and ecosystem functions.

5. The impact of antibiotics present in the aquatic environment on the frequency of resistance

transfer is questionable.



6. The present date suggests that the input of resistant bacteria into the environment from different

sources seems to be the most important source of resistance in the environment. Therefore, the

prudent use of antibiotics and disinfectants will significantly reduce the risk for the general

public and for the environment.

7. This not only means limiting the duration of selective pressure by reducing the treatment period

and the continuous use of sub-therapeutically concentrations, but also includes controlling the

dissemination of antibiotics being used, as well as prudent monitoring of resistance.

8. However, a full environmental risk assessment cannot be performed on the basis of the data

available; the availability of such data is a prerequisite if proper risk assessment and risk

management programs for both humans and the environment are to be undertaken. Therefore,

the careful use of antibiotics and the restriction of their input into the aquatic environment are

the matters of necessity.

IX. ACKNOWLEDGEMENT

Financial support from UGC fellowship to Mr. Vishvas Hare for Ph.D. work is dully acknowledged.

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