Presentation 21 Karunasagar · oqnrgene conferring resistance to quinolones are found in marine bacteria like Shewanella algaeand Vibriospp. oCTX-M beta-lactamase is present in environmental

AMR in zoonotic pathogens associated with aquatic environment

Iddya [email protected]

FMM/RAS/298: Strengthening capacities, policies and national action plans on prudent and responsible use of antimicrobials in fisheries Workshop 2

in cooperation with Malaysia Department of Fisheries and INFOFISH7-9 August 2017, Kuala Lumpur, Malaysia

Hazards associated with antimicrobial resistance in aquaculture

o Selection of resistance in pathogens of aquatic animals will impact health management in aquaculture.

o Antibiotic resistance genes selected in aquatic bacteria may spread to pathogens of aquatic animals or to zoonotic bacteria in the aquatic environment.

o Selection of resistance in zoonotic pathogens associated with aquatic environments.

Zoonotic bacteria in aquatic environment

o Autochthonous aquatic bacteria – eg Vibrio vulnificus, V. parahaemolyticus, Aeromonas hydrophila.

o Allochthonous bacteria entering aquatic environment through contamination with sewage, animal farm waste, hospital effluents – eg Salmonella enterica (various serovars)

Some truths and myths about antimicrobial resistance

o Antimicrobial resistance is a major problem in medical field. Infections with multidrug resistant bacteria are on the increase. (Truth)

o Indiscriminate use of antibiotics in medical, livestock and aquaculture sectors have contributed to selection and spread of antimicrobial resistance. (Truth)

o Antibiotic resistance emerged only due to anthropogenic use of antibiotics. (Myth)

o If antibiotic resistant bacteria are found in any farmed fish or aquaculture environment, this suggests that antibiotics are used in aquaculture. (Myth)

Antimicrobial resistance is ancient, natural and is found in environments with no exposure to antibiotics

o Viable multidrug-resistant bacteria have been cultured from the Lechuguilla Cave in New Mexico, totally isolated for >4 million years (Bhullar et al., 2012).

o Antibiotic resistant marine bacteria have been found as far as 522KM offshore and in deep sea at depths of 8200m (Aminov, 2011).

o Evolution of antibiotic resistance genes predates evolution of Actinomycetes.

o Some of the antibiotic resistance genes have not evolved to protect against antibiotics but have other metabolic functions.

Resistance genes have other functions in the cell

o ampC beta-lactamase is involved in maintaining normal morphology in Escherichia coli.

o Efflux pumps are involved in efflux of several compounds

o blaoxy beta-lactamase has metabolic function in Klebsiella oxytoca

Resistance genes found in environmental bacteria without exposure to antibiotics

o qnr gene conferring resistance to quinolones are found in marine bacteria like Shewanella algae and Vibrio spp.

o CTX-M beta-lactamase is present in environmental bacteria like Kluyvera

Some bacteria have intrinsic resistance

o The intrinsic resistance of a bacterial species to a particular antibiotic is the ability to resist the action of that antibiotic as a result of inherent structural or functional characteristics.

o For example, the glycopeptide antibiotic vancomycin inhibits peptidoglycan crosslinking by binding to target d-Ala-d-Ala peptides, but in Gram-negative organisms, it cannot cross the outer membrane and access these peptides in the periplasm

o Aeromonas has intrinsic beta-lactamases which makes them resistant to ampicillin.

Acquired antimicrobial resistance

o When microorganisms once sensitive to an antimicrobial agent become resistance to that particular antibiotic, the resistance is acquired.

o The acquired resistance could be due to genetic changes such as mutations or acquisition of genes contributing to resistance through horizontal gene transfer.

Acquired antimicrobial resistance

o Antibiotic resistance genes may be transferred through in mobile genetic elements such as plasmids, transposons, bacteriophages, genomic islands or integrons.

o Though integrons are not self-mobile, they contain gene cassettes that are mobile.

they harbor. Regardless of this lag inawareness, however, those integronswere certainly part of the first multi-drug resistance outbreaks in the 1950s.One proof is that Tn21, an integron-containing transposon, was involved inthe resistance phenotype propagated byplasmid NR1 (R100) in the very firstevents in Japan.

In light of the six-year time scale forthe emergence of multidrug-resistantShigella strains in Japan in 1956, it washardly disputable that bacteria were al-ready equipped with appropriate ge-netic tools for meeting the challenge ofmultidrug assaults. Indeed, molecularstudies during the last 15 years showhow powerful the bacterial integron re-combination machinery is, despite itsrelative functional simplicity.

Thus, all known integrons are com-posed of three essential elements forprocuring exogenous genes: (i) a genecoding for an integrase (intI), (ii) a pri-mary recombination site (attI), and (iii)a strong promoter (Pc). Integron inte-grases recombine, in a recA-indepen-dent manner, discrete units of circular-ized DNA known as gene cassettesdownstream of the resident Pc pro-moter at the proximal attI site, permit-ting expression of their encoded pro-teins (Fig. 1). Even considering onlythose that differ in nucleotide sequenceby more than 5%, more than 70 differ-ent resistance cassettes have been described, andthey confer resistance to all beta-lactams, allaminoglycosides, chloramphenicol, tri-methoprim, streptothricin, rifampin, erythro-mycin, and antiseptics of the quaternary ammo-nium compound family.

Moreover, all these integron-inserted cas-settes share several specific structural character-istics. For instance, the boundaries of each inte-grated cassette are defined by two GTTRRRY(core-site) sequences in the same orientation,which are the targets of the recombination pro-cess. The integrated cassettes also generally in-clude a single gene and an imperfect invertedrepeat located at the 3! end of the gene called anattC site (or “59-base element”), a diverse familyof sequences that function as recognition sitesfor the site-specific integrase. The attC sites vary

from 57 bp to 141 bp in length, and their nucle-otide sequence similarities are primarily re-stricted to the inverse core-site and the core-site(Fig. 2).

Three classes of integrons (RI) involved inmultidrug resistance expression were defined onthe basis of homology of their integrase genes.Each class appears capable of sharing and ac-quiring the same gene cassettes. Several cassettesappear to belong in two different classes ofintegrons, and the class 1 integrase can recom-bine several structurally diverse attC sites. TheIntI integrases belong to the catalytic family ofthe tyrosine (Y) recombinases that are involvedin the movement of numerous phages throughsite-specific recombination (such as the lambdaphage integrase, "Int) or in fundamental cellularprocesses such as chromosome dimer resolutionin cell division (XerC/D). In spite of these rela-

F I G U R E 1

Integron-mediated gene capture and the model for cassette exchange. Outline of theprocess by which circular antibiotic resistance gene cassettes (antR) are repeatedlyinserted at the specific attI site in a class1 integron downstream of the strong promoterPC. intI1, integrase encoding gene; Int, integrase IntI1.

Volume 70, Number 11, 2004 / ASM News Y 521

Phenotypic resistance and mechanisms of resistance

o When same phenotypic resistance is detected in two isolates eg one from aquatic environment and another from a clinical case, the two isolates may have different resistance genes.

o Eg tetracycline resistance could be due to (a) over production of efflux proteins or (b) production of ribosomal protection proteins or (c) production of tetracycline inactivating proteins

Antibiotic resistance in Vibrio vulnificus

o US CDC recommends a treatment course of doxycycline (100 mg PO/IV twice a day for 7-14 days) and a third-generation cephalosporin (e.g.,ceftazidime 1–2 g IV/IM every eight hours) or single therapy with fluroquinolones.

o trimethoprim-sulfamethoxazole in combination with an aminoglycoside is recommended for the treatment of pregnant women and children

o So far, therapeutic failures in V. vulnificus infections due to drug resistance has not been reported.

o Environmental strains tested in US and Europe are generally sensitive to antibiotics of therapeutic importance.

Antibiotic resistance in Vibrio vulnificus

o Some environmental isolates from South Africa showed resistance to cephalothin.

o Most Vibrio vulnificus strains isolated from Dutch eel farms showed resistance to cefoxitin, though this antibiotic was not used in eel aquaculture (Haenen et al., 2014).

Antibiotic resistance found in zoonotic Vibrio spp

o Vibrio parahaemolyticus: Resistance reported from different geographical regions

o Studies done in Korea show that all V. parahaemolyticus isolated from oysters were resistant to ampicillin and vancomycin and half the number of isolates exhibited resistance to cephalothin, rifampin and streptomycin (Kang et al., 2016).

o Studies in China with isolates from crustaceans and shellfish show much higher (over 90%) resistance to rifampin and 78% resistance to streptomycin (Hu and Chen, 2016)

FAO Fisheries and Aquaculture Report No. 937 FIPM/R937 (En) ISSN 2070-6987

Report of the

FAO EXPERT WORKSHOP ON THE APPLICATION OF BIOSECURITY MEASURES TO CONTROL SALMONELLA CONTAMINATION IN SUSTAINABLE AQUACULTURE Mangalore, India, 19–21 January 2010

FAO Fisheries and Aquaculture Report No. 937 FIPM/R937 (En) ISSN 2070-6987

Report of the

FAO EXPERT WORKSHOP ON THE APPLICATION OF BIOSECURITY MEASURES TO CONTROL SALMONELLA CONTAMINATION IN SUSTAINABLE AQUACULTURE Mangalore, India, 19–21 January 2010

FAO Fisheries and Aquaculture Report No. 937 FIPM/R937 (En) ISSN 2070-6987

Report of the

FAO EXPERT WORKSHOP ON THE APPLICATION OF BIOSECURITY MEASURES TO CONTROL SALMONELLA CONTAMINATION IN SUSTAINABLE AQUACULTURE Mangalore, India, 19–21 January 2010

Public health risk due to Salmonella in aquaculture

o Although Salmonella is a major foodborne pathogen, the products of aquaculture are rarely implicated in outbreaks.

o There are a number of pathways through which Salmonella can enter aquaculture environments. These include wild animals, domestic stocks, sewage contamination, storm water etc.

o Serovars commonly found in raw aquaculture products are rarely involved in human infections.

17

Table 3. Dominant Salmonella serotypes associated with human illness and seafood / aquaculture environment Human illness associated global rank 2002a

Seafood associated rank occurrence 1990–1998b

Aquaculture environment

(not rank ordered) 2001–2003c

Enteritidis (1) Weltevreden (1) Weltevreden Typhimurium (2) Senftenberg (2) Paratyphi-B (predominantly biovar

Java) Newport (3) Lexington (3) Senftenberg Heidelberg (4) Paratyphi-B (4)

(predominantly biovar Java)

Houten

Infantis (5) Enteritidis (5) Abaetetuba Hadar (6) Newport (6) Derby Virchow (7) Thompson (7) Aberdeen Javiana (8) Lanka (8) Javiana Saintpaul (9) Virchow (9) Hvittingfoos Montevideo (10) Hvittingfoss (10) Give Paratyphi B (16) Typhimurium (12) Newport Weltevreden (20) Derby(14) a Galanis et al., 2006 bHeinitz et al., 2000 c Data from: Hatha et al., 2003; Koonse et al., 2005; Kumar et al., 2009; Norhana et al., 2010

4.4 Salmonellosis and aquaculture products

There is very little specific data on the incidence of salmonellosis associated with aquaculture products. Epidemiological records of outbreaks associated with such food may be included under a general seafood category (even if they are freshwater products) or as fish or shellfish (possibly broken down into crustaceans and bivalve molluscs). Table 4 shows the incidence of salmonellosis associated with all food vehicles, and with seafood, for the European Union in 2007 (EFSA, 2009). Table 5 shows similar data for the United States (Lynch et al., 1996). The general level of gastroenteritis and salmonellosis is much higher in developing countries. However, none of the outbreak data available for developing countries was subdivided by food category and thus cannot be presented in an analogous manner. Table 6 shows the overall 10 most common serovars isolated from humans, as well as from various foods, in Thailand between 1993 and 2002 (Bangtrakulnonth et al., 2004).

The data for the European Union and the United States suggests that seafood is not a significant vehicle of salmonellosis in developed countries. It can therefore be inferred that aquaculture products, as a subset of this food category, is also not a significant vehicle. Other food products predominate with regard to the transmission of salmonellosis.

Salmonella has been isolated from aquaculture systems in both developing countries and developed countries. Studies done in Southeast Asia indicate that 16.1 percent of shrimp and 22.2 percent of water/mud samples were positive for Salmonella (Reilly and Twiddy, 1992). In US freshwater catfish ponds a prevalence of 5 percent (Wyatt et al., 1979) was observed and from eel culture ponds in Japan, a prevalence of 21 percent (Saheki et al., 1989) has been reported. Salmonella has also been isolated from pond water in a trout farm in Spain (Cesar-Javier et al., 1999). A relatively high percentage of 33 percent in US catfish and 50 percent in Vietnamese catfish were reported to be positive for Salmonella by Pal and Marshall (2009) and this may be due to the methodology used for isolation. But these data show that low prevalence of Salmonella can be seen in aquaculture systems in all parts of the globe.

10

Table 2. Salmonella detection in the aquatic environment Country(No of

samples) Type of sample

Positive sample

(%) Serotypes Resistance

(%) Reference

Spain

(5384)

Molluscs 3 Serotypes (N=20): Senftenberg 42.5% Typhimurium 15% Agona 9.4%

9 Martinez-Urtaza et al., 2004 Seawater 2.5

Morocco (801)

Mussels 10 Serotypes (N=3): Blockley 43.8% Kentucky 29.8% Senftenberg 26.3%

49.1 Setti et al., 2009

Sediments 6.8

Seawater 4.1

Mexico, Ensenada (1331)

Wastewater 16.2 Serotypes (N=20): Typhimurium 23.4% Vejle 6.2% Suberu 4.7%

– Simental et al., 2008

Stream water 10.6 Molluscs 7.4 Seawater 2.3

Mexico, Culiacan (138)

Water 80.4

Serotypes (N=29): Oranienburg 24,3% Saintpaul 9.0% Minnesota 6.3%

50.4

Jimenez, Chaidez and Martinez-Urtaza, personal communication

Asian countries (1234)

Shrimps 1.6

Weltevreden Paratyphi B Abaetetuba

– Koonse et al., 2005

Holding pond water 2.5

Pond sediments 1.0 Pond grow-out water 3.5

Source water 5.0 Source sediment 24

Viet Nam (50) Shellfish 18.0 - 11.1 Van et al.,

2007

India, Cochin (443)

Fish 30.5 Serotypes (N=30): Weltevreden 8.2% Rissen 7.8% Typhimurium 6.7%

82% Kumar et al., 2008

Shrimps 29

Clams 34.1

The vast majority of studies looking at the presence of Salmonella in aquatic and marine environments have evidenced two main observations: only a small but constant number of serovars have been found in these environments and, in most cases, these do not coincide with the main zoonotic serovars identi ed in the surrounding areas (Catalao Dionisio et al., 2000; Heinitz et al., 2000; Martinez-Urtaza et al., 2004; Polo et al., 1999; Venkateswaran et al., 1989; Wilson and Moore, 1996). In spite of the variability in sampling size (n= 37 to 251), in most of these studies the maximum number of serotypes identi ed has been around 20 (Catalao Dionisio et al., 2000; Martinez-Urtaza et al., 2004; Venkateswaran et al., 1989; Wilson and Moore, 1996). Serovar Typhimurium has been shown to be the most common clinically signi cant serovar isolated from environmental samples in many parts of the world (Baudart et al., 2000; Catalao Dionisio et al., 2000; Martinez-Urtaza et al., 2004; Polo et al., 1999; Willson and Moore, 1996; Simental and Martinez-Urtaza, 2008), which attests to its capacity of adaptation and survival in external environments (Baudart et al., 2000). Salmonella Senftenberg has been recognized one of the major serotypes identified in marine environments and raw seafood worldwide. It has been one of the predominant serovars detected in the coastal waters of Portugal (Catalao Dionisio et al., 2000), in crustaceans from India (Hatha and Lakshmanaperumalsamy, 1997), in raw seafood imported into the United States especially from tropical countries (Heinitz et al., 2000),

10

Table 2. Salmonella detection in the aquatic environment Country(No of

samples) Type of sample

Positive sample

(%) Serotypes Resistance

(%) Reference

Spain

(5384)

Molluscs 3 Serotypes (N=20): Senftenberg 42.5% Typhimurium 15% Agona 9.4%

9 Martinez-Urtaza et al., 2004 Seawater 2.5

Morocco (801)

Mussels 10 Serotypes (N=3): Blockley 43.8% Kentucky 29.8% Senftenberg 26.3%

49.1 Setti et al., 2009

Sediments 6.8

Seawater 4.1

Mexico, Ensenada (1331)

Wastewater 16.2 Serotypes (N=20): Typhimurium 23.4% Vejle 6.2% Suberu 4.7%

– Simental et al., 2008

Stream water 10.6 Molluscs 7.4 Seawater 2.3

Mexico, Culiacan (138)

Water 80.4

Serotypes (N=29): Oranienburg 24,3% Saintpaul 9.0% Minnesota 6.3%

50.4

Jimenez, Chaidez and Martinez-Urtaza, personal communication

Asian countries (1234)

Shrimps 1.6

Weltevreden Paratyphi B Abaetetuba

– Koonse et al., 2005

Holding pond water 2.5

Pond sediments 1.0 Pond grow-out water 3.5

Source water 5.0 Source sediment 24

Viet Nam (50) Shellfish 18.0 - 11.1 Van et al.,

2007

India, Cochin (443)

Fish 30.5 Serotypes (N=30): Weltevreden 8.2% Rissen 7.8% Typhimurium 6.7%

82% Kumar et al., 2008

Shrimps 29

Clams 34.1

The vast majority of studies looking at the presence of Salmonella in aquatic and marine environments have evidenced two main observations: only a small but constant number of serovars have been found in these environments and, in most cases, these do not coincide with the main zoonotic serovars identi ed in the surrounding areas (Catalao Dionisio et al., 2000; Heinitz et al., 2000; Martinez-Urtaza et al., 2004; Polo et al., 1999; Venkateswaran et al., 1989; Wilson and Moore, 1996). In spite of the variability in sampling size (n= 37 to 251), in most of these studies the maximum number of serotypes identi ed has been around 20 (Catalao Dionisio et al., 2000; Martinez-Urtaza et al., 2004; Venkateswaran et al., 1989; Wilson and Moore, 1996). Serovar Typhimurium has been shown to be the most common clinically signi cant serovar isolated from environmental samples in many parts of the world (Baudart et al., 2000; Catalao Dionisio et al., 2000; Martinez-Urtaza et al., 2004; Polo et al., 1999; Willson and Moore, 1996; Simental and Martinez-Urtaza, 2008), which attests to its capacity of adaptation and survival in external environments (Baudart et al., 2000). Salmonella Senftenberg has been recognized one of the major serotypes identified in marine environments and raw seafood worldwide. It has been one of the predominant serovars detected in the coastal waters of Portugal (Catalao Dionisio et al., 2000), in crustaceans from India (Hatha and Lakshmanaperumalsamy, 1997), in raw seafood imported into the United States especially from tropical countries (Heinitz et al., 2000),

Public health risk due to Salmonella in aquaculture

o Clonal Salmonella Weltevreden isolated from geographically separated aquaculture sites in Vietnam.

o Does S. Weltevreden survive for long periods of time in aquatic environments?

o AMR in S. Weltevreden low frequency. Only two of 37 isolates, showed resistance to ampicillin and tetracycline and to streptomycin, sulfisoxazole and tetracycline, respectively (Ponse et al., 2008)

Summary

o Antimicrobial resistance is found in aquatic environment, even when there is no exposure to antibiotics.

o Some aquatic bacteria are of zoonotic importance, but AMR is still not a major concern in these.

o Zoonotic bacteria like Salmonella may reach aquaculture environments through different pathways

o AMR is not a major concern in serovars associated with aquaculture

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