since antibiotics were first discovered in 1928 , they have been widely used in human and animal medicine . however , most antibiotics are excreted and enter the soil or water environments through wastewater and fertilization with manure ( kmmerer , 2003 ) .
antibiotic contamination of the natural environment has been documented in many countries , including those of europe , north america , and east asia , and areas of antibiotic contamination are widespread throughout the region ( segura et al . , 2009 ) .
the pathways whereby residual antibiotics enter the environment are varied and include wastewater effluent discharge and runoff and leaching from land fertilized with agricultural or human waste .
an increasing number of reports show a rise in the occurrence and distribution of antibiotics in surface waters in europe ( hirsch et al . , 1999 ;
, 2001 ; kolpin et al . , 2002 ; kim and carlson , 2007 ) , and canada ( metcalfe et al . , 2004 ) .
cases of antibiotic contamination of wastewater are also well - known ( gbel et al . , 2005 ; and others ) .
some antibiotics can be found in ground water ( baquero et al . , 2008 ) . in china ,
antibiotics have been found in animal wastewater , pond water , animal farm effluents , and rivers .
the most frequently detected antibiotics in china include sulfamethazine ( 75% ) , oxytetracycline ( 64% ) , tetracycline ( 60% ) , sulfadiazine ( 55% ) , and sulfamethoxazole ( 51% ) , which were found at maximum concentrations of 211 , 72.9 , 10.3 , 17.0 , and 63.6  ppb , respectively ( wei et al . , 2011 ) .
in korea , environmental contamination with tetracyclines and sulfonamides was reported at the ppb level ( ok et al . , 2011 ) .
recent excellent reviews by kmmerer ( 2009a , b ) summarize contamination of the environment with antibiotics and the spread of antibiotic resistance . this manuscript will focus on indochina environment having unique situation as following .
unlike the socioeconomic - unified continents of europe and north america , asia is more geographically and culturally disjunct , and can not be readily grouped as a single geopolitical entity .
it therefore remains difficult to obtain a generalized theory of antibiotic and antibiotic resistance contamination across all asian countries .
this manuscript focuses specifically on the region of indochina and the major antibiotics in this area , including quinolones , sulfonamides , and tetracyclines .
countries of  indochina  include vietnam , thailand , cambodia , lao pdr , and myanmar .
of these , vietnam and thailand have particularly become a center of rapid industrial development and economic growth ; however this growth has far outpaced investment in public infrastructure , leading chemical pollution and sanitary issues .
although indochina is globally one of the most active regions of industrial growth and development , there remains few studies on antibiotic contamination relative to their western and east asian counterparts ( le and munekage , 2004 ; nhiem et al .
, 2008 ; zhang et al . , 2009 ; hoa et al . , 2011 ; takasu et al . ,
contamination levels are highly variable , ranging from scales of ppm to ppt ( le and munekage , 2004 ; managaki et al . , 2007 ;
, 2008 ; hoa et al . , 2011 ; takasu et al . , 2011 ) .
table 1 summarizes the various antibiotic contaminants that have been detected in indochina aquatic environments . until now for antibiotic resistance in this area , major reports consisted of studies on salmonella isolates ( ogasawara et al . , 2008 ; sirichote et al . , 2010 ; and others ) .
, not quantifiable due to overlapped interfering peak . large amounts of antibiotics are used for human medicine , livestock farming , and aquaculture in southeast asia .
in addition , we observed that antibiotics are used as additives in ice to prevent decay of harvested fish in fish markets .
it is believed that high concentrations of antibiotics applied in this manner will remain in the fish , and will then be taken in by people upon consumption .
such widespread antibiotic contamination of the environment and food supply likely promotes the development of antibiotic - resistant bacteria , both in the environment and within the human intestine .
prior to the early 2000s , few studies addressed antibiotic contamination in indochina ( le and munekage , 2004 ) , although the occurrence of antibiotic - resistant bacteria in the region was reported ( kim et al . , 2004 ; le et al . , 2005 ; zhang et al . , 2009
however , recent studies on drug contamination , combined with detection of antibiotic - resistant bacteria and/or resistance genes , have provided a clearer picture of the problem in this area of asia ( hoa et al . , 2011 ; takasu et al . ,
aquaculture farming system known as vac ( vegetation , aquaculture , and cage ) is common .
the vac system is a recycling farm , typically consisting of a vegetable field , an aquaculture pond , and caged animals , and has been practiced since the 1980s . in the vac system ,
livestock manure ( usually from pigs , chickens , and ducks ) is directly transported to fish ponds and to vegetable and rice fields . this untreated sewage and wastewater from the livestock operations is used for fish culture and for fertilization of the vegetable fields .
the animal manure contributes to the eutrophication of pond water , which enhances phytoplankton growth .
the vac system is considered a very economical method of recycling farming ( hop , 2003 ) ; however , the heavy use of antibiotics in livestock increases the prevalence of enteric antibiotic - resistant bacteria and the potential for antibiotic - resistant pathogenic bacteria to arise .
subsequent discharge of antibiotic residues in farm waste likely increases the number of antibiotic - resistant bacteria in the environment , which in turn promotes the further selection and transfer of antibiotic resistance genes within the microbial community in the surrounding environment ( petersen and dalsgaard , 2003 ; heuer and smalla , 2007 ) .
( 2011 ) reported that enteric bacteria of pigs in vac showed high resistance to nalidixic acid and enrofloxacin , which would be dispersed to water environment .
that is , the abundance of antibiotic - resistant bacteria and genes initially increases in the intestinal tracts of livestock as a result of exposure to high concentrations of various antibiotics .
next , horizontal gene transfer occurs among various bacteria in the environment surrounding the vac operation .
finally , the antibiotic resistance genes move to other environments via water use and the food supply .
the antibiotic resistance genes are brought together , exchanged , and spread in vac systems .
if the antibiotic - resistant bacteria come into contact with human pathogens and commensal bacteria , the risk to human health may increase significantly .
although there are many snapshot reports of antibiotic resistance ( reviewed in zhang et al . , 2009 ) , further dynamic study is needed to evaluate the risks posed by the presence of antibiotic resistance genes in the aquatic environments of developing countries in asia .
fluoroquinolones ( fqs ) are fully synthetic antibiotics that are widely used in humans , animals , and fish ( grave et al . , 1999 ; le and munekage , 2004 ; samanidou et al . , 2005 ;
the antibacterial mechanism of fqs is based on inhibition of bacterial dna gyrase or topoisomerase iv , which are enzymes essential for dna replication .
first - generation fqs are piromidic acid , oxolinic acid , and nalidixic acid , which were widely used in aquaculture in the 1970s in japan . in tropical asian countries
, oxolinic acid still appears to be one of the major drugs used ( grslund et al .
second - generation ( ciprofloxacin , cip and norfloxacin , nor ) and third - generation ( levofloxacin , lev and its enantiomer ofloxacin , ofl ) compounds are used in hospitals and animal husbandry in indochina ( takasu et al . ,
the fqs are photo - degradable , with a half - life in pure water of 105 and 90  min for nor and cip , respectively ( burhenne et al . , 1997 ) .
however , fqs in the environment are relatively stable in water and sediment ( kmmerer , 2004 ; le and munekage , 2004 ) , which might be due to sorption onto particulates ( nowara et al . , 1997 ) .
lai and lin ( 2009 ) reported that oxolinic acid and flumequine could be retained in sediment for 9.515 and 3.66.4  days , respectively .
such long half - lives in the environment pose a selective pressure for environmental bacteria .
although the bioavailability of antibiotics is suspected to decrease upon adsorption on clay and humic substances , no supporting evidence has been reported .
( 2003 ) reported that 74% of thailand shrimp aquaculture farmers use antibiotics , primarily nor . le and munekage ( 2004 )
reported detecting nor at 0.53.5  ppm in the water column and 2001500  ppm in the sediment of intensive ponds and improved extensive ponds in vietnam .
oxolinic acid could be detected in the water column at a concentration similar to that of nor , but not in the sediment .
the  water column concentration  indicates present inflow , while the  sediment concentration  indicates the value integrated over time ( takasu et al . , 2011 ) .
the presence of antibiotics in both samples suggests that the compounds being used at present and are retained in the sediment .
ciprofloxacin is commonly used with other drugs , such as griseofulvin , rifampicin , and oxytetracycline for shrimp larvae in vietnam ( thuy et al . , 2011 ) .
the most recent quantification of fqs in indochina aquatic environments showed that the average concentrations were higher in thailand ( ofl , 7400  ppb ; nor , 209  ppb ; cip , 328  ppb ; lom , 67.4  ppb ) than in vietnam ( ofl , 255  ppb ; nor , 41.1  ppb ; cip , 162  ppb ; lom , 25.3  ppb ; takasu et al .
both ofl and nor were confirmed as major environmental contaminants in both countries ; however , contamination at aquaculture sites was lower than at vac farms and city canals in both countries .
a recent decrease in drug application and/or dilution effects may explain the improved contamination situation in aquaculture settings .
since fqs are not natural compounds , it is believed that bacteria do not possess fq resistance genes .
several resistance mechanisms have been reported , including mutation of dna gyrase ( fukuda et al . , 1990 ) ,
impermeability to fqs due to loss of porins , and extrusion by overexpressed efflux pumps ( hirai et al . , 1986 , 1987 ) .
a number of plasmid - mediated quinolone resistance ( pmqr ) genes ( qnra , qnrb , and qnrs ) have also been identified ( robicsek et al . , 2006 ) .
these pmqr genes are transferred horizontally among bacteria and encode a protein of the pentapeptide repeat family that has been shown to block the actions of fqs on purified dna gyrase and topoisomerase iv ( robicsek et al . , 2006 ) .
the origin of these genes is thought to be chromosomes in aquatic bacteria ( poirel et al .
thus , the aquatic environment is hypothesized to be a natural reservoir of fq - resistant bacteria and resistance genes . as mentioned above , the environmental concentration of fqs
was found to be much higher in thailand than in vietnam . despite the lower level of contamination ,
the occurrence rate of fq - resistant bacteria was found to be higher in vietnam than thailand ( takasu et al . , 2011 ) .
( 2011 ) demonstrated that there is no relationship between the concentration of fqs in the environment and the rate of bacterial resistance .
kmmerer ( 2003 ) reported that exposure to a sub - inhibitory concentration induces the emergence of resistant bacteria in aquatic environments .
it was reported that fqs at concentrations 10100 times lower than the mic induce the expression of a functional gene ( herold et al . , 2005 ) ,
mutations ( gillespie et al . , 2005 ) , and morphological changes ( loubeyre et al . , 1993 ) at the single - cell level .
low concentrations are thus effective inducers of drug - resistant bacteria . the effect of disinfectants on bacteria is considerably reduced in the presence of organic matter ( kawamura - sato et al . , 2008 ) , suggesting that the actual active concentration in the environment might be lower than analysis suggests . the importance of low concentrations of fqs to the development of antibiotic resistance should not be ignored , and additional research involving both instrumental analyses and bioassays is needed to estimate the actual concentration of fqs acting upon environmental bacteria .
a broad range of bacteria can acquire resistance to fqs , including enteric bacteria ( escherichia coli ) , pathogenic bacteria ( e.g. , acinetobacter ) , and aquatic bacteria ( e.g. , brevundimonus ) .
proteobacteria and actinobacteria are the major taxa of fq - resistant bacteria ( takasu et al . , 2011 ) , indicating that fq - resistant bacteria are not limited to specific groups .
it was hypothesized that bacteria from humans and animals as well as natural aquatic bacteria are potential reservoirs of fq resistance genes .
a number of resistance genes have been identified in fq - resistant bacteria , including qnra , qnrb , qnrs , and aac(6)-ib - cr ( takasu et al . , 2011 ) . since qnra , qnrb , and qnrs
are thought to have originated from the chromosomes of water - dwelling bacteria ( poirel et al .
, 2005a , b ) , it is possible that these genes can be transferred from aquatic bacteria to human bacteria ( hernndez et al . , 2011 ) .
to date , pmqr genes including aac ( 6)-ib - cr , which are linked with other drug resistance genes and are transferable , have only been discovered in gammaproteobacteria such as vibrio ( poirel et al .
, 2005a ) , shewanella ( poirel et al . , 2005b ) , and aeromonas ( cattoir et al . , 2008 ; picao et al . ,
the number of fq - resistant salmonella enterica isolates found in thailand increased in the late 1990s , and the resistant isolates possessed the same gyra region , suggesting rapid spread of the resistant bacteria ( hakanen et al . , 2001 ) .
the gyra mutation is located on the chromosome , but recent research indicates that gyra can also be transferred ( ferrandiz et al .
spreading of both resistant bacteria and chromosomal resistance genes might be a cause of the observed widespread drug resistance in asia .
however , precisely how fq resistance genes are spread among various environments is not known , although mutation - based resistance and transferable genes should be considered .
the dynamics of fq resistance genes are of interest and importance from the viewpoints of human clinical and gene evolution studies .
sulfonamides , which are also synthetic antibiotics , have been widely used to treat bacterial and protozoan infections in humans , domestic animals , and aquaculture species since their introduction to clinical practice in 1935 ( perreten and boerlin , 2003 ; le and munekage , 2004 ; blahna et al . , 2006 ) .
sulfonamides inhibit folate biosynthesis by competing with the natural substrate p - amino - benzoic acid for binding to dihydropteroate synthase ( dhps ) , an enzyme in the folic acid synthesis pathway .
while the use of sulfonamides in humans has decreased in developed countries , they are still frequently used in developing asian countries due to their low cost ( macrolides are 13 $ /tablet , whereas sulfonamides are 2 cent / tablet in vietnam ) .
sulfamethoxazole ( smx ) , a commonly used sulfonamide analog , was detected at ppm levels in shrimp ponds in vietnam in the late 1990s ( le and munekage , 2004 ) .
although such levels are extremely high , smx levels in rivers in japan and vietnam are typically around 100  ppt in recent years ( managaki et al . , 2007 ) .
this was confirmed in various locations in vietnam ( hoa et al . , 2011 ) , where concentrations were in the 6004000  ppt range throughout the year in a city canal .
trimethoprim was detected in the 201800  ppt range as well , probably because it is often used with smx . in livestock farm wastewater ,
the concentration of smx was lower , in the 68600  ppt range , whereas that of sulfamethazine was much higher ( 4006000  ppt ) .
other sulfonamides were either not detected or found at trace levels , suggesting that smx and sulfamethazine are the major sulfonamides in use in vietnam at present .
since sulfonamides inhibit the formation of dihydrofolic acid ( perreten and boerlin , 2003 ) , bacterial resistance to sulfonamides can occur through mutations in the chromosomal dhps gene ( folp ) or through acquisition of an alternative dhps gene ( sul ) whose product has a low affinity for sulfonamides ( perreten and boerlin , 2003 ) .
acquisition of sulfonamide resistance through sul genes is the most prevalent mechanism ( enne et al .
sulfonamide resistance is globally prevalent among human and animal pathogens ( kerrn et al . , 2002 ; perreten and boerlin , 2003 ; antunes et al . , 2005 ) ; however , the presence of sul genes is not equally distributed among bacterial populations ( kerrn et al . , 2002 ; antunes et al . , 2005 ; hammerum et al . ,
the sul2 gene , which is related to class i integron , is common in acinetobacter isolated from fish farms and chicken manure in thailand ( agers and petersen , 2007 ) .
however , hoa et al . ( 2008 ) reported that sul1 was the major sul gene among smx - resistant bacteria isolated from vac farms , city canals , and aquaculture sites in vietnam .
most of these reports were based on isolated bacteria , which may bias interpretation of results due to the processes of isolation ; therefore , we can not obtain an accurate assessment of the distribution of antibiotic resistance genes from culturable bacteria alone .
a direct quantification approach indicated that sul1 is the major sulfonamide resistance gene in lagoon and river waters in the usa ( pruden et al . , 2006 ) .
it is likely that such non - culture monitoring of antibiotic resistance genes will increase in the near future ( for example , pei et al . , 2006 ) .
a culture - dependent study of smx - resistant bacteria revealed that acinetobacter is one of the major reservoirs of sul genes in asian aquatic environments ( agers and petersen , 2007 ; hoa et al . , 2011 ) . since acinetobacter , especially a. baumannii ,
is known to be an important opportunistic pathogen with multi - drug resistance ( vila and pachn , 2011 ) , the risk posed by environmental a. baumannii to human health should be examined further .
low concentration of smx can affect on bacterial nitrate metabolism and select bacterial species ( underwood et al . , 2011 ) , suggesting importance of trace contamination by smx .
tetracyclines are a family of broad - spectrum antibiotics that includes tetracycline , oxytetracycline ( otc ) , chlortetracycline , doxycycline , and minocycline ( chopra and roberts , 2001 ) .
these antibiotics inhibit protein synthesis in gram - positive and gram - negative bacteria by preventing the binding of aminoacyl - trna molecules to the 30s ribosomal subunit ( geigenmuller and nierhaus , 1986 ; ross et al . , 1998 ) .
owing to their broad - spectrum activity and low toxicity , tetracyclines are used in the treatment of a number of human skin and dental diseases .
tetracyclines are also used in agriculture as growth promoters in farm animals and are used widely as prophylaxes in plant agriculture and aquaculture around the world ( chopra and roberts , 2001 ) , including southeast asia ( thuy et al . , 2011 ) .
the first - generation tetracyclines include otc , chlorotetracycline , and 6-demethylchlorotetracycloine , which were developed in the 1940s .
minocycline and doxycycline were launched as second - generation drugs in the 1960s ( thaker et al . , 2010 ) .
a new glycylcycline derivative , tigecycline , which is active against most otc - resistant bacteria , acinetobacter , mrsa , and extended - spectrum -lactamase ( esbl ) producers , was licensed in 2005 ( livermore , 2005 ) . despite the variety of available analogs ,
otc is still commonly used in animal production and aquaculture ( holmstrm et al . , 2003 ;
nonaka et al . , 2007 ; and others ) , although use as growth promoters has been banned in eu until 2006 ( castanon , 2007 ) . as tetracycline - resistant bacteria , high occurrence of tetracycline - resistant salmonella from human ( 50% ) and animals ( pig 34% and poultry 76% ) are reported in vietnam ( vo et al . , 2010 ) , as well as enterococcus and e. coli ( both 80% < ; dang et al . , 2011 ) .
similar results are known in thailand , e.g. , chicken ( 42% ) and chicken meat ( 45% ) , and pork ( 11% ) and pork meat ( 20% ; ogasawara et al . , 2008 ) . tetracycline resistance genes , tet series , from the resistant bacteria have been well - studied ( roberts , 2005 ; and others ) .
studies have indicated that some tet genes are persistent in aquaculture site without selective pressure ( tamminen et al . , 2011 ) , pristine environments and in animals ( gilliver et al . , 1999 ; rahman et al .
tracking of tet genes in aquatic environments , including pristine areas , has been examined in the usa ( pruden et al . , 2006 ;
storteboom et al . , 2010a , b ) , japan ( kim et al . , 2004 ; nonaka et al . , 2007 ) , and other countries ( zhang and zhang , 2011 ) ; however , similar research in indochina aquatic environments is limited ( kobayashi et al . , 2007a ; suzuki et al . , 2008 )
monitoring and phylogenetic analyses are needed if we are to obtain a thorough understanding of the origin and spread dynamics of tet genes .
kobayashi et al . ( 2007a ) detected tet(m ) , tet(s ) , and tet(w ) genes throughout the mekong river delta .
they found that tet(s ) and tet(w ) have only one genotype each , while tet(m ) has at least two genotypes .
one tet(m ) genotype is identical to the gene encoded in various plasmids and transposons of gram - positive and gram - negative bacteria , and another is similar to a gene encoded in tn1545 of enterococcus faecalis ( 99% identity in pcr product , 170  bp/171  bp ) . since the two types could be found in the same sample , it was assumed that more than one source of the gene exists .
this hypothesis was partly confirmed by microbial diversity analysis using denatured gradient gel electrophoresis analysis , which indicated a positive relationship between the shannon index ( h ) value and the tet gene detection- and otc - resistant bacteria occurrence - rates across a wide area , ranging from the mekong river and tonle sap lake , cambodia to the south china sea ( suzuki et al . , 2008 ) .
gene exchange and horizontal transfer among various bacterial species in natural environments have been demonstrated ( smets and barkay , 2005 ) .
ribosomal protection protein ( rpp ) genes , including tet(m ) , tet(s ) , and tet(w ) , are transferred among bacteria ( chopra and roberts , 2001 ) , and this phenomenon was experimentally confirmed using combinations of marine bacteria ( gene donors ) and e. coli ( neela et al . , 2009 ) .
( 2007b ) , which holds that the rpp genes were derived through duplication and divergence of an ancient gtpase before the divergence of the three domains .
the rpps may have originally provided ribosomal protection against other chemical substances in the environment .
it is known that exposing microbial assemblages to one toxicant can result in indirect selection for bacteria with resistance to multiple , chemically unrelated toxicants ( baker - austin et al . , 2006 ; baker et al . , 2006 ) .
for example , exposure to cd , ni , ampicillin , and tetracycline significantly increases the frequency of bacterial resistance to multiple , chemically unrelated metals and antibiotics ( stepanauskas et al . , 2006 ) .
metals and antibiotics are known not only for being associated to similar efflux pumps , but also for inducing co - resistance ( baker - austin et al .
aquatic environments in indochina are highly contaminated with many kinds of persistent organic pollutants , metals ( suzuki and takada , 2009 ) , and antibiotics ( managaki et al .
, environmental bacteria can acquire drug resistance naturally over time as well as through horizontal gene transfer .
both transferable paralogous resistance genes and canonical resistance genes might be present in natural environments . from the viewpoint of both drug resistance and
evolutionary biology , rpp genes are thus of considerable interest ( kobayashi et al . , 2007b ) .
indochina aquatic environments are particularly vulnerable to the development of antibiotic - resistant bacteria due to pollution with antibiotics and other chemicals in the absence of adequate wastewater treatment systems .
recent important findings concerning antibiotic resistance are as follows : ( 1 ) sub - lethal concentrations of antibiotics accelerate the development of antibiotic resistance ( yeh et al . , 2009 ; gullberg et al . ,
2010 ) , and ( 2 ) chemicals other than antibiotics can promote development of antibiotic resistance ( alonso et al .
both of these scenarios are suspected to occur in natural environments , especially in asian countries .
correlation between the number of antibiotic drugs detected and the occurrence of multi - drug - resistant bacteria in vietnam was found ( hoa et al . , 2011 ) , indicating that multi - drug contamination is almost certain to result in an increase in the prevalence of multi - drug - resistant bacteria in aquatic environments .
it is important to note that contamination with various antibiotics correlates strongly with induction of multi - drug resistance , even if the contaminant concentration is low .
multi - drug resistance is a serious issue , particularly in asia , and is likely to become of even greater concern in the future .
furthermore , contamination of antibiotics with trace concentrations can alter microbial ecosystem in terms of metabolism and diversity ( underwood et al . , 2011 ) , indicating the importance to consider unexpectedly effect than antimicrobial resistance . there is still a considerable gap in knowledge regarding the environmental chemistry and microbiology in the environment .
the authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest .
 southeast asia has become the center of rapid industrial development and economic growth . 
 however , this growth has far outpaced investment in public infrastructure , leading to the unregulated release of many pollutants , including wastewater - related contaminants such as antibiotics . 
 antibiotics are of major concern because they can easily be released into the environment from numerous sources , and can subsequently induce development of antibiotic - resistant bacteria . 
 recent studies have shown that for some categories of drugs this source - to - environment antibiotic resistance relationship is more complex . 
 this review summarizes current understanding regarding the presence of quinolones , sulfonamides , and tetracyclines in aquatic environments of indochina and the prevalence of bacteria resistant to them . 
 several noteworthy findings are discussed : ( 1 ) quinolone contamination and the occurrence of quinolone resistance are not correlated ; ( 2 ) occurrence of the sul sulfonamide resistance gene varies geographically ; and ( 3 ) microbial diversity might be related to the rate of oxytetracycline resistance . 
Introduction
Why Indochina?  Specific Situation of Indochina
Quinolone Resistance
Sulfonamide Resistance
Tetracycline Resistance
Conclusion
Conflict of Interest Statement