Antimicrobial residues, non-typhoidal Salmonella, Vibrio spp. and associated microbiological hazards in retail shrimps purchased in Ho Chi Minh city (Vietnam).

We investigated antimicrobial residues, non-typhoidal Salmonella (NTS), Vibrio spp. and their associated antimicrobial resistance (AMR), in shrimps locally purchased in Ho Chi Minh City (Vietnam). In addition, we investigated the relationship between AMR in NTS, Vibrio spp. and antimicrobial residue in the same sample. A total of 40 samples of shrimp heads/shells from different retail sources was cultured using ISO 6579-1:2017 (NTS) and ISO/TS 21872-1:2007 (Vibrio spp.). Phenotypic antimicrobial susceptibility was investigated using Vitek (NTS, 34 antimicrobials) and disk diffusion (Vibrio spp., 12 antimicrobials). A total of 9 (22.5%) samples contained antimicrobial residue, including tetracyclines, fluoroquinolones, sulfonamides and macrolides (in 7.5%, 7.5%, 2.5% and 2.5% of samples, respectively). Shrimp samples from supermarkets had a higher prevalence of antimicrobial residue than those purchased in street markets (50% vs. 13.3%) (p = 0.049). A total of 30 (75%) samples were contaminated with NTS. All samples contained Vibrio spp., with V. parahaemolyticus being most common (87.5% samples). A total of 58.9% NTS isolates were multidrug resistant. With regards to the highest priority, critically important antimicrobials, the highest resistance corresponded to quinolones (14.4-47.8%), followed by 3rd and 4th generation cephalosporins (3.3-7.8%). Vibrio spp. isolates were characterised by their high resistance against ampicillin (82.7%) and 3rd generation cephalosporins (8.3-16.5%). Extended Spectrum Beta-Lactamase (ESBL) activity was detected in 28.1% V. parahaemolyticus isolates. Half of ESBL-positive V. parahaemolyticus strains harboured bla CTX-M1. We found an association between the presence of residues and the number of resistances for NTS (p = 0.075) and Vibrio spp. isolates (p = 0.093) from the same sample. These findings suggest that the presence of residues may contribute to the selection of AMR in foodborne pathogens in shrimps. Authorities should strengthen policies aiming at restricting inappropriate antimicrobial usage in shrimp farming, and step up monitoring of antimicrobial residues and food-borne pathogens at retail in Vietnam.

Introduction

Antimicrobial resistance (AMR) is one of the greatest threats to our society (O'Neill, 2016). Among other sources, humans may acquire AMR bacterial infections or AMR-encoding genes through the consumption of contaminated food, including fish and shellfish (Cabello et al., 2013, Likotrafiti et al., 2018). In recent years shrimp farming has rapidly increased, reaching a global production of 3.2 million metric tons in 2017, much of it taking place in Asia (Anon., 2018). This increase is happening in a context of rapid globalization of markets, as well as the threat of climate change (Lauria et al., 2018). Antimicrobials are widely used in shrimp and aquaculture production, both to treat and prevent diseases (Henriksson et al., 2018). Contamination of aquaculture food products with antimicrobial residues represents a potential health hazard to the consumer due to food poisoning, the development of allergy problems, changes of the intestinal flora, as well as the emergence and subsequent spread of antimicrobial resistance (Okocha, Olatoye, & Adedeji, 2018).

Non-typhoidal Salmonella (NTS) and certain Vibrio spp. are major microbiological hazards associated with shrimp and seafood consumption (Baker-Austin et al., 2018, Tusevljak et al., 2012). Vibrio parahaemolyticus is the leading cause of seafood-borne bacterial gastroenteritis in the world. Both its thermostable direct hemolysin (tdh) and tdh-related hemolysin (trh) are considered major virulence factors of this micro-organism (Raghunath, 2015). In the late 1990s’, V. parahaemolyticus was implicated in a large outbreak of enteric disease in central Vietnam, with 523 cases reported (Chowdhury et al., 2004).

NTS is a major cause of gastroenteritis worldwide (Majowicz et al., 2010). In Vietnam, NTS is recognised as a major cause of pediatric diarrhoea (Thompson et al., 2012). There is also evidence of an increase in the incidence of severe invasive infections in hospitalised patients associated due to this organism (Lan et al., 2016, Nga et al., 2012).

The Vietnamese shrimp industry has experienced a considerably expansion over recent years, with most of its production being aimed the export market (mostly to the USA, Europe and Japan). In 2017, shrimp exports made up about half of the total Vietnam seafood exports, with sales worth 3.8 billion US$ (Hong, Hien, Thu, & Lebailly, 2017).

Shrimp exports are regularly screened for their microbiological safety by the companies themselves. However, little is known about the microbiological safety of shrimps available for domestic consumption. Therefore, the aims of this study were: (1) to investigate major foodborne hazards associated with shrimps from local retail sites in Ho Chi Minh City (HCMC), Vietnam, such as antimicrobial residues, NTS and Vibrio spp.; and (2) to characterise the AMR profile of these organisms, including the presence of Extended Spectrum Beta-Lactamases (ESBL) and colistin resistance. In addition we investigated the relationship between the presence of AMR in the two bacterial species and antimicrobial residues in the same batches, which to our knowledge has not been previously investigated.

Methods

Sample collection and identification

Batches of shrimps (250–300 g each) were purchased from 40 different retail sites located in 10 districts of HCMC (Vietnam) from March to June 2018. In order to maximize the diversity of sources, from each district three street markets and one supermarket were selected. From each retail site, one batch of live or dead shrimps (chilled, not frozen) was purchased. Shrimps were collected into a clean plastic bag, and were transported to the laboratory within 2 h in an ice-containing box. Five representative specimens per batch were weighted using precision scales. Shrimp species were identified based on their morphological features. Using a pair of sterile scissors, the heads, legs and exoskeleton were separated from the muscle tissue, and were subsequently pooled (shell mix). Muscle tissue samples were investigated for the presence of antimicrobial residues, and the shell mixes were investigated for NTS and Vibrio spp.

Antimicrobial residue analyses

Shrimp muscle tissue samples were investigated for antimicrobial residues using a hierarchical approach. Firstly, they were screened using PremiTest (R-Biopharm AG, Germany), an assay based on the inhibition growth of Bacillus stearothermophilus spores. Positive or inconclusive result samples were then examined for the presence of macrolides, amphenicols, tetracyclines, β-lactams and sulfonamides antimicrobial classes, as well as for the presence of chloramphenicol, streptomycin and gentamicin/neomycin using a Charm II analyzer 7600 (Charm Sciences, USA) (Gaudin, Juhel-Gaugain, Moretain, & Sanders, 2008). Samples that tested positive by Charm II were then confirmed for specific antimicrobials within each class by Ultra-High Performance Liquid Chromatography Tandem Mass Spectrometry (LC-MS/MS). In addition, PremiTest-positive samples were investigated for quinolones by LC-MS/MS. (See Table A with the list of antimicrobials investigated by LC-MS/MS).

Isolation of NTS and vibrio spp.

The shrimp shell mixes were investigated for NTS using a modified ISO 6579–1:2017 method. Briefly, from each sample 25 g of homogenized shell mix was pre-enriched in 225 mL buffered peptone water (BPW, Oxoid, UK) at 37 °C for 18 h. A loop of pre-enrichment media was then inoculated on Modified Semi-solid Rappaport-Vassiliadis (MSRV, Oxoid, UK), and incubated at 41.5 °C for 24 h. and positive growth was further inoculated on chromogenic Rambach agar (CHROMagar, France) and incubated at 37 °C for 24 h (Carrique-Mas, Barnes, McLaren, & Davies, 2009). Matrix-Assisted Laser Desorption Ionization Time-of-Flight Mass Spectrometry (MALDI-TOF MS) (Bruker, Germany) was used to investigate the species identity of three suspected (pink) isolates from each culture. NTS isolates were further classified as either group B, C, D, E or ‘others’ according to the Kauffmann-White scheme using relevant poly-O antiserum (Grimont P.A. & Weill, 2007). Shrimp shell mixes (25 g) were also investigated for the presence of Vibrio spp. using a modification of the ISO/TS 21872–1:2007 method. Briefly, the steps were: (1) 25 g of the shell mix was suspended in 225 mL of alkaline saline peptone water (ASPW) at 41.5 °C for 24 h; (2) a loop of enrichment was cultured on thiosulfate citrate bile and sucrose agar (TCBS, Oxoid, UK) at 37 °C for 24 h. Four suspected Vibrio spp. isolates from each sample were confirmed by MALDI-TOF.

Antimicrobial susceptibility testing

All confirmed NTS isolates were tested for their antimicrobial susceptibility against a panel of 34 antimicrobials belonging to 11 classes by Vitek (bioMérieux, Marcy l’Etoile, France) (Livermorea et al., 2002) (33 antimicrobials), as well as by Etest (BioMérieux, France) (colistin). All Vibrio spp. isolates were tested using the disk diffusion method for 12 antimicrobials representative of eight classes (Oxoid, UK). The full list of antimicrobials investigated is displayed in Tables C4 and C5. NTS and Vibrio spp. isolates were classified as susceptible, intermediate or resistant according to CLSI guidelines (M100-S27 for NTS, M45-A2 for Vibrio spp.) (Anon, 2010, Anon, 2017a). A strain was defined as ‘multidrug resistant’ (MDR) if it was fully resistant to antimicrobials belonging to at least three different classes. The potential production of ESBLs was investigated by the ‘comparative disk diffusion method’, using cefotaxime and ceftazidime disks alone, as well as in combination with clavulanate (Anon., 2017a). Antimicrobial susceptibility results were sorted according to the WHO list of antimicrobials of human health importance (Anon., 2017b).

Determination of serovar identity of NTS

NTS isolates were further classified as belonging to either group B, C, D, E or ‘others’ according to the Kauffmann-White scheme using poly-O antiserum (Grimont P.A. & Weill, 2007). From each sample, one isolate representative of each a serogroup-antimicrobial susceptibility testing result pattern was investigated by Multi-Locus Sequence Typing (MLST). The MLST scheme followed is based on seven loci aroC, dnaN, hemD, hisD, purE, sucA and thrA (Yun et al., 2015).

Investigation of tdh and trh genes of vibrio spp. by PCR

The presence of genes encoding a thermostable direct hemolysin (tdh) and a tdh-related hemolysin gene (trh) was investigated by PCR in all Vibrio spp. isolates (Tada et al., 1992). Positive and negative control isolates were used. The positive control isolates originated from confirmed human cases.

Investigation of ESBL and plasmid mediated colistin resistance-encoding genes by PCR

The presence of blaCTX-M(1, 2, 8, 9 and 25), blaTEM, blaSHV, and blaOXA genes (all encoding extended-spectrum β-lactamases) was investigated by multiplex PCR (Dallenne, Da Costa, Decre, Favier, & Arlet, 2010) in all NTS and Vibrio spp. isolates that tested positive phenotypically for ESBL. The presence of plasmid-mediated genes (mcr-1 to mcr-5) among phenotypic colistin-resistant isolates was investigated by multiplex PCR (Rebelo et al., 2018).

Statistical analyses

We investigated 40 shrimp batches with the aim of determining the prevalence of residues, based on an expected prevalence of 8%, a 95% level of confidence and an 8% relative precision. We expected to obtain 50 NTS isolates from these batches. This sample size (50) allowed determining a prevalence of MDR of ∼27% (based on published data) with a 95% level of confidence and a 9% relative precision. The prevalence of contamination and resistance across variables was compared using chi-square tests. The level of agreement between presence of residues and presence of intermediate (or resistant) isolates in each shrimp batch sample was investigated using the kappa statistic.

Results

Shrimp samples

The 40 batches investigated included specimens of five shrimp species: white leg shrimp (Litopenaeus vannamei) (30) (22.5 g weight; 16.2 ± Standard Deviation (SD) 1.2 cm length), giant tiger shrimp (Penaeus monodon) (5) (22.0 g; 15.4 ± SD 1.4 cm), banana shrimp (Penaeus merguiensis) (3) (17.3 g, 12.5 ± SD 1.6 cm), greasy-back shrimp (Metapenaeus ensis) (1) (11.5 g; 11.5 ± SD 1.4 cm), and giant prawn (Macrobrachium rosenbergii) (34.3 g; 16.6 ± SD 1.1 cm). The descriptive data for the samples investigated, and the prevalence of PremiTest, NTS and Vibrio spp. sample positivity results are shown in Table 1.

Table 1

Description, prevalence of residues, NTS and Vibrio spp. among 40 shrimp batches purchased in HCMC.

Variable	No. samples	No. (%) positive
		PremiTest	NTS (%)	Vibrio species
				V. parahaemolyticus	V. navarrensis	V. alginolyticus	V. cholerae non-O1	V. vulnificus	V. fluvialis
Type of retail site
Supermarket	10	5 (50.0%)	6 (60.0%)	9 (90.0%)	7 (70.0%)	4 (40%)	6 (60.0%)	2 (20.0%)	0 (0%)
Street market	30	4 (13.3%)	24 (80.0%)	26 (86.7%)	17 (56.7%)	17 (56.7%)	9 (30.0%)	7 (23.3%)	4 (13.3%)
Shrimp species
White leg shrimp	30	8 (26.7%)	22 (73.3%)	26 (86.7%)	15 (50.0%)	16 (53.3%)	12 (40.0%)	7 (23.3%)	4 (13.3%)
Giant tiger shrimp	5	1 (20.0%)	3 (60.0%)	4 (80.0%)	5 (100%)	2 (40.0%)	2 (40.0%)	1 (20.0%)	0 (0%)
Other species	5	0 (0%)	5 (100%)	5 (100%)	4 (80%)	3 (60%)	1 (20%)	1 (20%)	0 (0%)
Condition
Alive	17	3 (17.6%)	12 (70.6%)	16 (94.1%)	8 (47.1%)	11 (64.7%)	4 (23.5%)	5 (29.4%)	4 (23.5%)
Dead	23	6 (26.1%)	18 (78.3%)	19 (82.6%)	16 (69.6%)	10 (43.5%)	11 (47.8%)	4 (17.4%)	0 (0%)
Retail price (per kg)
≤170 k VNDa	22	6 (27.3%)	14 (63.6%)	18 (81.8%)	14 (63.6%)	12 (54.5%)	9 (40.9%)	5 (22.7%)	1 (4.5%)
>170 k VND	18	3 (16.7%)	16 (88.9%)	17 (94.4%)	10 (55.6%)	9 (50%)	6 (33.3%)	4 (22.2%)	3 (16.7%)
Total	40	9 (22.5%)	30 (75.0%)	35 (87.5%)	24 (60.0%)	21 (52.5%)	15 (37.5%)	9 (22.5%)	4 (10.0%)

VND=Vietnam Dong (1USD = 23 kVND).

Antimicrobial residues

Antimicrobial residues were detected in 9/40 (22.5%) samples by PremiTest. Shrimp samples from supermarkets had a higher prevalence of antimicrobial residues than those purchased in street markets (50% vs. 13.3%) (χ2 = 3.871, p = 0.049). Four of the nine PremiTest-positive samples were positive by Charm II, whereas the remaining five tested negative (Table 2). Tetracyclines, sulfonamides, and macrolides were detected by Charm II in 7.5%, 2.5%, and 2.5% of samples, respectively. Antimicrobials identified by LC-MS/MS included two tetracycline antimicrobials (tetracycline and oxytetracycline) and two fluoroquinolone antimicrobials (ciprofloxacin and flumequine). The Charm II macrolide-positive sample tested negative for both tylosin and erythromycin by LC-MS/MS. Two samples contained antimicrobial residues above the maximum residue limits (MRL) according to Vietnamese regulations (one contained tetracycline and sulfamethoxazole; another oxytetracycline, and ciprofloxacin). Oxytetracycline and flumequine were also found in two other samples although at concentrations below the MRL (43.7 μg/kg and 41.2 μg/kg, respectively).

Table 2

Results of antimicrobial residue testing by Charm II and LC-MS/MS among 9 shrimp samples that tested positive by PremiTest.

Sample ID	Description	Charm II (antimicrobial class)	LC-MS/MS (antimicrobial)	Concentration of antimicrobial active ingredient (μg/kg)	MRL (μg/kg)
1	White leg shrimp, dead	Tetracyclines	Tetracycline	590.7	100
		Sulfonamides	Sulfamethoxazole	157.6	100
			Flumequinea	38.5	200
2	White leg shrimp, dead	Tetracyclines	Oxytetracycline	122.2	100
			Ciprofloxacin*	30	Not allowed
3	White leg shrimp, dead	Macrolides	ND	–	–
			Flumequinea	41.2	200
4	White leg shrimp, dead	Tetracyclines	Oxytetracycline	43.7	100
5	White leg shrimp, dead	ND	ND	–	–
6	White leg shrimp, live	ND	ND	–	–
7	White leg shrimp, live	ND	ND	–	–
8	White leg shrimp, live	ND	ND	–	–
9	Tiger shrimp, dead	ND	ND	–	–

Highest priority, critically important antimicrobial; ND=Not detected; MRL = Maximum Residue Limits according to Vietnamese regulation.

Prevalence of contamination with NTS serovars and vibrio spp.

A total of 90 NTS isolates were recovered from 30/40 (75%) shrimp batches (Table 1). The prevalence of NTS among samples purchased in street markets was higher than that from supermarket samples, although this difference was not significant (80% vs. 60%; χ2 = 0.711; p = 0.399). There was lower probability of recovering NTS from PremiTest-positive samples than from PremiTest-negative samples (5/9 vs. 25/31), although this difference was not significant (Fisher’s exact test, p = 0.190). MLST was performed on 62 isolates with a unique serogroup-antimicrobial susceptibility pattern. The remaining 28 isolates were assigned to serovar based on MLST results of isolates recovered from the same sample and with the same serogroup-AST pattern. A total of 28 MLST sequence types (ST) corresponding to 25 NTS serovars were identified. One NTS strain (Group B) could not be assigned to ST, and therefore its serovar identity was not determined (Table B). The most prevalent serovars identified were Braenderup (present in 20% samples), Anatum (16.7% samples), Saintpaul (13.3% samples), Rissen and Litchfield (10% samples each). All (100%) samples were positive for Vibrio species, yielding 133 isolates. Among six Vibrio species, V. parahaemolyticus was the most common species (87.5% samples), followed by V. navarrensis (60%), V. alginolyticus (52.5%), V. cholerae non-O1 (37.5%), V. vulnificus (22.5%) and V. fluvialis (10%) (Table 1).

Antimicrobial susceptibility among NTSisolates

Among highest priority-critically important antimicrobial classes, the highest prevalence of resistance corresponded to quinolones (nalidixic acid, ciprofloxacin, ofloxacin, levofloxacin, moxifloxacin) (range 14.4–47.8%), followed by 3rd and 4th generation cephalosporins (cefixime, cefotaxime, ceftazidime and ceftriazone) (3.3–7.8%). Among high priority-critically important antimicrobials, resistance was highest against aminoglycosides (16.7% gentamicin and 7.8% tobramycin), monobactams (7.8% aztreonam), and glycylcyclines (3.3% tigecycline) (Fig. 1, Table C). A total of 58.9% isolates were MDR. The highest prevalence of MDR corresponded to Group B isolates (76.2%; 95% CI 58.0–94.4%), followed by Group D (75.0; 95% CI 32.6–100%) and Group C (51.4%; 95% 35.2–67.5%). Seven isolates (7.8%) (from 3 samples) were identified as ESBL-positive. They were identified as serovars Infantis (3), Give (3) and Braenderup (1). The isolates identified as Infantis and Give (three of each, from two different samples) had identical antimicrobial susceptibility profile.

Fig. 1

Phenotypic resistance of NTS isolates by group. Pale bars indicate the percent of isolates showing intermediate resistance; dark bars indicate percent of isolates with full resistance. 95% binomial confidence intervals have been drawn around the percentage of resistant plus intermediate resistant isolates.

Antimicrobial resistance of vibrio spp. isolates

Results of antimicrobial susceptibility testing of 133 of Vibrio spp. against 12 antimicrobial drugs are shown in Fig. 2 and in Table D. The highest prevalence of resistance corresponded to ampicillin (82.7%), followed by co-trimoxazole (18.8%) and 3rd generation cephalosporins (16.5% cefotaxime; , 8.3% ceftazidime). All (100%) V. parahemolyticus and V. alginolyticus isolates were fully resistant to ampicillin. The prevalence of resistance against amoxicillin-clavulanic, penems, aminoglycosides, tetracyclines, quinolones and phenicols was <11.3% in all cases. A total of 18/64 (28.1%) V. parahaemolyticus were ESBL producers; however none (0%) of the 69 non-V. parahaemolyticus strains were ESBL producers. Overall, 18 of 133 (13.5%) Vibrio spp. isolates were MDR, but this percent was 28.1% among V. parahemolyticus, and 0% among other Vibrio species.

Fig. 2

Phenotypic resistance of Vibrio spp. isolates. Pale bars indicate the percent of isolates showing intermediate resistance; dark bars indicate percent of isolates with full resistance. 95% binomial confidence intervals have been drawn around the percentage of resistant plus intermediate resistant isolates.

Detection of toxin-encoding genes

None of the 133 Vibro spp. isolates tested positive for either the tdh or trh genes.

Detection of ESBL genes in NTS and vibrio spp. isolates

The phenotypically ESBL-positive serovar Branderup isolate tested negative for all ESBL genes investigated. All three serovar Infantis isolates (from the same sample) were positive for blaCTX-M9. The three serovar Give isolates tested positive for both blaCTX-M1 and blaTEM. Interestingly, all isolates were fully susceptible to cefepime and cefoxitin. A total of 9/18 (50%) ESBL-positive V. parahaemolyticus strains were positive for blaCTX-M1. In addition, one of these isolates tested positive to the blaTEM gene.

Relationship between residues, and AMR in NTS and vibrio spp. isolates

NTS isolates from shrimp samples that tested positive to PremiTest (n = 15) were resistant to a median of 10 antimicrobials [IQR 3–13], whereas NTS isolates from samples testing negative (n = 75) were resistant to 5 antimicrobials [IQR 0–9] (Wilcoxon test W = 724.5, p = 0.075). Vibrio spp. isolates from PremiTest-positive samples (n = 29) were resistant to a median of 1 [IQR 1–4] antimicrobial, compared with 1 [IQR 1–2] among Vibrio spp. from PremiTest-negative samples (n = 104) (Wilcoxon = 1792; p = 0.093) (Fig. 3). We found fair agreement between presence of residue (PremiTest) and presence of co-trimoxazole and ciprofloxacin resistant NTS from the same sample (kappa values 0.265 and 0.365; p ≤ 0.016). We also found a fair agreement between presence of a quinolone residue and ciprofloxacin resistance in Vibrio spp. isolates from the same sample (kappa = 0.383; p = 0.005). In addition, there was a moderate agreement between samples that contained ESBL-positive Vibrio spp. and ESBL-positive NTS isolates (kappa = 0.515, p < 0.001) (Table D). However, in neither of the two samples that contained both phenotypically ESBL-positive NTS and Vibrio spp. we could demonstrate the presence of the same genes: in one sample V. parahaemolyticus was positive for both blaTEM and blaCTX-M1, whereas no ESBL genes were detected in the NTS isolate; in the other, NTS harboured the blaCTX-M9, wheras V. parahaemolyticus tested negative for all ESBL genes.

Fig. 3

Number of phenotypic resistances among NTS and Vibrio spp. isolates from PremiTest-positive and PremiTest-negative shrimp samples.

Discussion

This study evidenced a high prevalence of contamination of shrimp samples with antimicrobial residues (22.5%), NTS (75%), and Vibrio spp. (100%). This result is in line with a previous survey of shrimps from local markets in the Red River and Mekong Delta regions of Vietnam (13.0% and 33.3%, respectively) (Pham et al., 2015), but higher than previous results reported from the Vietnamese provinces of HCMC, Thai Binh and Nha Trang (8.8, 1.8 and 3.2%, respectively) (Uchida et al., 2016). However, we were only able to establish the identity of the antimicrobial residue in 4 of 9 samples that tested positive by a bacterial inhibition test. This may be the result of a false-positive result in our screening test, or (more likely) due to the presence of antimicrobial residues not investigated in this study. All antimicrobials confirmed in our samples (tetracyclines, ciprofloxacin, flumequine) are known to be commonly used in Vietnamese aquaculture (Pham et al., 2015), although levels of flumequine (two samples) and oxytetracycline (one sample) where within the legal limits. We found considerable agreement between the screening results by Charm II and the residues detected by LC-MS/MS. The exception was a macrolide-positive sample by Charm that subsequently tested negative for both erythromycin and tylosin. We hypothesize that this may reflect the use of spiramycin, another macrolide antimicrobial known to be used in aquaculture (Sekkim & Kum, 2011) that was not investigated. It is likely that the observed prevalence of residues in shrimp samples reflects the use of antimicrobials late in the production cycle, thought to be common practice in Vietnamese aquaculture (Pham et al., 2015). A surprising finding was the higher prevalence of residues in shrimps procured from supermarkets than from (more informal) street markets. This suggests that intensive production systems (normally associated with the supermarket supply chains) may be associated with a higher levels of antimicrobial usage. This is consistent with a previous study that reported a higher prevalence of residues in shrimp products procured in supermarkets compared with wholesale markets (Uchida et al., 2016). The observed prevalence of contamination with NTS in this study (75%), is higher than in previous studies from Vietnam, China, Thailand and Bangladesh (prevalence levels ranging between 13% and 55%) (Minami et al., 2010, Phan et al., 2005, Pinu et al., 2007, Uddin et al., 2015). A recent study investigating NTS in 25 g meat samples purchased across markets in Vietnam resulted in a comparatively lower prevalence (71.8% in chicken, 70.7% in pork and 62.2% in beef samples) (Nhung et al., 2018). In contrast with the antimicrobial residue results, shrimp samples purchased in street markets had higher prevalence of NTS contamination than shrimps purchased in supermarkets, although this difference was not statistically significant. We hypothesize that this may reflect deficiencies in the cold chain associated with street markets. A study in Thailand reported absence of NTS in shrimps purchased in supermarkets, compared with a 50.2% prevalence among shrimps purchased in street markets (Minami et al., 2010).

We found a considerable serovar diversity (25) of NTS in our samples, with serovars Braenderup, Anatum, Saintpaul and Rissen accounting for 46.7% of all NTS isolates. Most serovars detected in shrimps have been isolated in outbreaks of human disease in different countries. A study from 2004 on 56 human clinical (diarrhoea, fever) in Vietnam identified 12 different serovars (Vo et al., 2006), of which four (Anatum, Albany, Typhimurium and Enteritidis) were detected in our shrimp samples. However, in that study, serovars Typhimurium and Enteritidis accounted for 50% of human cases, whereas in our study they only accounted for 6.6% of all isolates. A more recent study on invasive (bloodstream infection) isolates identified 19 serovars, of which 8 were identified in our study (Lan et al., 2016). Surprisingly, serovar Weltevreden, which was predominant in a previous study on shrimp farms in the Mekong Delta of Vietnam (Uddin et al., 2015) was found only in one sample, and was a pansusceptible strain. A possible explanation for this discrepancy is that NTS contamination of shrimps mainly occurs after harvesting. It has been shown experimentally that seafood is an excellent nutrient media supporting vigorous post-harvest NTS multiplication at ambient temperatures (Kumar, Datta, & Lalitha, 2015). Since no routine diagnostic or surveillance data exists for human salmonellosis, it is not possible to know to what extent shrimps are a significant source of NTS to the community in Vietnam. A total of 58.9% NTS isolates were MDR, a figure higher compared with overall MDR prevalence in isolates from meat in a previous study using the same antimicrobial panel (52.2%) (Nhung et al., 2018). Overall resistance to ciprofloxacin (33.3%) is of great concern, since this antimicrobial is often used to treat enteric infections. We found that 7.8% isolates were ESBL-producers and were fully resistant to at least one of the four 3rd-4th generation cephalosporins investigated. ESBL-producing NTS organisms were identified as belonging to serovars Braenderup (1), Infantis (3), and Give (3). These ESBL-producing isolates were MDR (including quinolone resistance), although were susceptible to carbapenems. The latter is the first choice drug in the treatment of ESBL-producing microorganisms (Zhanel et al., 2007).

All (100%) retail shrimp samples were contaminated with Vibrio spp., being V. parahaemolyticus the most prevalent species (87.5% samples). Other Vibrio species were isolated in 10.0–60.0% samples. These levels of contamination are comparable with studies in northern Vietnam (99.5% prevalence) (Tra et al., 2016) and Malaysia (100%) (Letchumanan, Yin, Lee, & Chan, 2015), although higher than results from Turkey (67%) (Mus, Cetinkaya, & Celik, 2014), confirming that Vibrio spp. organisms are omnipresent in the shrimp farm aquatic environment (Gopal et al., 2005). In addition to V. parahaemolyticus, both V. vulnificus and V. cholerae non-O1 are also known to cause severe human disease (Deshayes et al., 2015). We did not, however, find evidence of any of the two major virulence genes investigated (tdh and trh) in any of the 133 Vibrio spp. isolates. Previous research in Malaysia has shown a low prevalence of tdh (4%) and trh (12%) genes in non-clinical V. parahaemolyticus isolates (Paydar, Teh, & Thong, 2013). In a study on 47 environmental isolates from India, only 4.2% and 2.1% harboured the tdh and trh genes, respectively (Koralage et al., 2012). However there was no evidence of these genes in isolates investigated in northern Vietnam (Tra et al., 2016), Hong Kong (Wong, Liu, Wan, & Chen, 2012), or Sri Lanka (Koralage et al., 2012). In the late 1990s’, V. parahaemolyticus was implicated in a large outbreak of enteric disease in central Vietnam, with 548 cases reported (Tuyet et al., 2002). It was determined in further analyses that the prevailing serovar changed over time (O3:K6 in 1997, O4:K68 in 1998, O1:K25 in 1998–1999), and that 85% clinical isolates harboured either the tdh or trh genes (Chowdhury et al., 2004).

We found that 82.7% of Vibrio spp. isolates were resistant to ampicillin (100% for V. parahaemolyticus and V. alginolyticus). This prevalence was comparable to published resistance levels among V. parahaemolyticus isolates from shrimps in northern Vietnam (87%) and Malaysia (82%) (Letchumanan et al., 2015, Tra et al., 2016). A total of 31.3% V. parahaemolyticus isolates were resistant to third generation cephalosporins and 28.1% were ESBL producers. In about half of those strains, the gene carried was blaCTX-M1. However, in another half the molecular basis for ESBL activity could not be established. Given that these genes are highly mobile, and are often inserted in plasmids and transposons, we hypothesize that V. parahaemolyticus may act as a reservoir of ESBL genes (Canton, Jose, & Galan, 2012). Interestingly we found an association between the presence of ESBL in NTS and V. parahaemolyticus isolated from the same sample.

The study confirmed the association between presence of antimicrobial residues and phenotypic resistance in NTS and Vibrio spp. in the same samples. This may be a reflection of AMR selection during the farming process, or may alternatively reflect post-harvesting contamination. The latter would be likely if the antimicrobial residue in the sample contributed to preferentially select for contamination with more resistant strains.

This study provides evidence of high levels of contamination with antimicrobial residues, NTS and Vibrio spp. among shrimps purchased in retail sites in HCMC. We found a high prevalence of MDR among NTS, with worryingly high levels of quinolone resistance. Although most Vibrio spp. isolates are unlikely to be pathogenic, the high carriage levels of ESBL in V. parahaemolyticus is of concern. We recommend authorities to enforce existing policies aiming at restricting inappropriate antimicrobial usage on shrimp farms, stepping up hygiene conditions during harvesting, transporting and retailing of shrimps, and to establish monitoring of antimicrobial residues, NTS and Vibrio spp. (focused on ESBL and virulence factors) in Vietnam. These findings should encourage the establishment of microbiological surveillance systems focused on health hazards in aquaculture food products, as well as strengthening laboratory capacity to enable comparisons between NTS and Vibrio spp. isolates from shrimps and human cases of disease.

Conflicts of interest

The authors declare no conflict of interest.

Table A

Panel of antimicrobial residues tested by LC-MS/MS.

WHO category	Antimicrobial class	Antimicrobial
Highest priority-critically important	Fluoroquinolones	Enrofloxacin, ciprofloxacin, norfloxacin, flumequine
	Macrolides	Tylosin, erythromycin
Highly important	Tetracyclines	Tetracycline, oxytetracycline, chlortetracycline
	Sulfonamides	Sulfadiazine, sulfamethazine, sulfaquinoxaline, sulfamethoxazole

Table B

Serovar identity of 62 NTS isolates investigated by MLST.

No.	Serogroup	Serovar	Sequence type (ST)	Number of isolates
1	B	Saintpaul	50	3
	B	Saintpaul	27	2
2	B	Derby	40	4
3	B	Paratyphi B	42	2
4	B	Typhimurium	36	2
5	B	Agona	13	1
6	B	Stanley	29	1
7	B	Unknown	Unknown	1
8	C	Braenderup	311	6
	C	Braenderup	22	3
9	C	Rissen	469	6
10	C	Bovismorbificans	1499	1
11	C	Infantis	32	1
12	C	Litchfield	214	3
13	C	Ohio	329	1
14	C	Albany	292	1
15	C	Bareilly	203	1
16	C	Kentucky	314	1
17	C	Potsdam	2039	1
18	D	Enteritidis	11	4
19	E	Anatum	64	8
20	E	Give	516	1
	E	Give	831	1
21	E	Senftenberg	14	1
22	E	Weltevreden	365	1
23	E	London	155	1
24	Other	Poona	1069	1
25	Other	Kedougou	1543	2
26	Other	Urbana	512	1

Table C.1

Phenotypic antimicrobial susceptibility results for 90 NTS isolates from shrimps, listed by antimicrobial class ranked by their importance according to the WHO classification. The figures correspond to the number of intermediate resistant, followed by the number of fully resistant isolates.

Class and antimicrobial	Group B (n = 21)
	Agona	Derby	Paratyphi B	Saintpaul	Stanley	Typhimurium	Unknown	Intermediate resistant (%)	Fully resistant (%)
No. isolates	1	4	3	9	1	2	1
Samples (n = 30) (%)	1 (3.3%)	2 (6.7%)	1 (3.3%)	5 (16.7%)	1 (3.3%)	2 (6.7%)	1 (3.3%)
Highest priority-critically important
Cephalosporins (3rd & 4th gen.)
Cefepime	0│0	0│0	0│0	0│0	0│0	0│0	0│0	0(0%)	0(0%)
Cefixime	0│0	0│0	0│0	0│0	0│0	0│0	0│0	0(0%)	0(0%)
Cefotaxime	0│0	0│0	0│0	0│0	0│0	0│0	0│0	0(0%)	0(0%)
Ceftazidime	0│0	0│0	0│0	0│0	0│0	0│0	0│0	0(0%)	0(0%)
Ceftriazone	0│0	0│0	0│0	0│0	0│0	0│0	0│0	0(0%)	0(0%)
Polyymyxins
Colistin	0│0	0│0	0│0	0│0	0│0	0│0	0│0	0(0%)	0(0%)
Quinolones
Ciprofloxacin	1│0	4│0	2│0	0│9	0│1	1│0	0│1	8(38.1%)	11(52.4%)
Levofloxacin	1│0	4│0	0│0	3│6	1│0	0│0	0│1	9(42.9%)	7(33.3%)
Moxifloxacin	0│0	0│0	0│0	0│6	1│0	0│0	0│1	1(4.8%)	7(33.3%)
Nalidixic Acid	0│0	0│0	0│0	0│6	0│0	0│0	0│1	0 (0%)	7(33.3%)
Ofloxacin	0│1	0│4	0│0	0│9	0│1	0│0	0│1	0 (0%)	16(76.2%)
High priority-critically important
Aminoglycosides
Amikacin	0│0	0│0	0│0	0│0	0│0	0│0	0│0	0 (0%)	0 (0%)
Gentamicin	0│0	0│0	0│0	0│6	0│0	0│0	0│1	0 (0%)	7(33.3%)
Tobramycin	0│0	0│0	0│0	7│0	0│0	0│0	0│0	7(33.3%)	0 (0%)
Carbapenems and other penems
Ertapenem	0│0	0│0	0│0	0│0	0│0	0│0	0│0	0(0%)	0(0%)
Imipenem	0│0	0│0	0│0	0│0	0│0	0│0	0│0	0(0%)	0(0%)
Meropenem	0│0	0│0	0│0	0│0	0│0	0│0	0│0	0(0%)	0(0%)
Glycylcyclines
Tigecycline	0│0	0│0	0│0	3│0	0│0	0│0	0│0	3(14.3%)	0(0%)
Monobactams
Aztreonam	0│0	0│0	0│0	0│0	0│0	0│0	0│0	0 (0%)	0 (0%)
Penicillins
Ampicillin	0│1	0│4	0│0	0│9	0│1	0│0	0│1	0(0%)	16(76.2%)
Piperacillin	0│1	0│4	0│0	0│9	0│1	0│0	0│1	0 (0%)	16(76.2%)
Ticarcillin	0│1	0│4	0│0	0│9	0│1	0│0	1│0	1(4.8%)	15(71.4%)
Highly important
Amphenicols
Chloramphenicol	0│0	0│4	0│0	0│9	0│0	0│0	0│1	0(0%)	14(66.7%)
Cephalosporins (1st and 2nd gen.)
Cefalotin	0│0	1│0	0│0	0│0	0│0	0│0	0│0	1(4.8%)	0(0%)
Cefoxitin	0│0	0│0	0│0	0│0	0│0	0│0	0│0	0 (0%)	0(0%)
Cefuroxime	0│0	0│0	0│0	3│0	0│0	0│0	0│0	3(14.3%)	0(0%)
Cefuroxime Axetil	0│0	0│0	0│0	3│0	0│0	0│0	0│0	3(14.3%)	0(0%)
Tetracyclines
Minocycline	0│1	0│4	1│0	0│9	0│1	2│0	0│1	3(14.3%)	16(76.2%)
Tetracycline	0│1	0│4	0│0	0│9	0│1	0│0	1│0	1 (4.8%)	15(71.4%)
Other
Penicillin & β-lactamase inhibitor
Amoxicillin/Clavulanic Acid	0│0	0│0	0│0	1│0	0│0	0│0	0│0	1(4.8%)	0(0%)
Piperacillin/Tazobactam	0│0	0│0	0│0	0│0	0│0	0│0	0│0	0(0%)	0(0%)
Folate pathway inhibitors
Trimethoprim	0│1	0│1	0│0	0│8	0│1	0│0	0│0	0(0%)	11(52.4%)
Folate pathway inhibitors/Sulfonamides
Trimethoprim/Sulfamethoxazole	0│1	0│1	0│0	0│9	0│1	0│0	0│1	0(0%)	13(61.9%)
Nitrofurantoins
Nitrofurantoin	0│0	0│0	0│0	0│0	0│0	0│0	0│0	0(0%)	0(0%)
ESBL (%)	–	–	–	–	–	–	–	–	0(0%)
MDR (%)	1(100%)	4(100%)	–	9(100%)	1(100%)	–	1(100%)	–	16(76.2%)

Table C.2

Class and antimicrobial	Group C (n = 37)
	Albany	Bareilly	Bovismor-bificans	Braenderup	Infantis	Kentucky	Litchfield	Ohio	Potsdam	Rissen	Intermediate resistant (%)	Fully resistant (%)
No. isolates	1	1	3	13	3	1	3	2	1	9
Samples (n = 30) (%)	1 (3.3%)	1 (3.3%)	1 (3.3%)	6 (20%)	1 (3.3%)	1 (3.3%)	3 (10%)	1 (3.3%)	1 (3.3%)	3 (10%)
Highest priority-critically important
Cephalosporins (3rd & 4th gen.)
Cefepime	0│0	0│0	0│0	0│0	0│0	0│0	0│0	0│0	0│0	0│0	0(0%)	0(0%)
Cefixime	0│0	0│0	0│0	0│1	0│3	0│0	0│0	0│0	0│0	0│0	0(0%)	4(10.8%)
Cefotaxime	0│0	0│0	0│0	0│1	0│3	0│0	0│0	0│0	0│0	0│0	0(0%)	4(10.8%)
Ceftazidime	0│0	0│0	0│0	0│0	0│0	0│0	0│0	0│0	0│0	0│0	0(0%)	0(0%)
Ceftriazone	0│0	0│0	0│0	0│1	0│3	0│0	0│0	0│0	0│0	0│0	0(0%)	4(10.8%)
Polyymyxins
Colistin	0│0	0│0	0│0	0│0	0│0	0│0	0│0	0│0	0│0	0│0	0 (0%)	0(0%)
Quinolones
Ciprofloxacin	1│0	0│1	0│0	2│8	0│3	1│0	1│2	0│0	0│1	4│0	9(24.3%)	15(40.5%)
Levofloxacin	1│0	1│0	0│0	10│0	3│0	0│0	2│1	0│0	0│1	3│1	20(54.1%)	3(8.1%)
Moxifloxacin	0│0	0│0	0│0	1│0	0│0	0│0	1│1	0│0	0│1	0│0	2 (5.4%)	2(5.4%)
Nalidixic Acid	0│1	0│0	0│0	0│2	0│3	0│0	0│1	0│0	0│1	0│0	0(0%)	8(21.6%)
Ofloxacin	0│1	0│1	0│0	0│10	0│3	0│0	0│3	0│0	0│1	1│3	1(2.7%)	22 (59.5%)
High priority-critically important
Aminoglycosides
Amikacin	0│0	0│0	0│0	0│0	0│0	0│0	0│0	0│0	0│0	0│0	0(0%)	0 (0%)
Gentamicin	0│0	0│0	0│0	0│1	0│3	0│0	0│0	0│0	0│1	0│0	0(0%)	5(13.5%)
Tobramycin	0│0	1│0	0│0	0│1	0│3	0│0	1│0	0│0	1│0	0│0	3(8.1%)	4 (10.8%)
Carbapenems and other penems
Ertapenem	0│0	0│0	0│0	0│0	0│0	0│0	0│0	0│0	0│0	0│0	0(0%)	0(0%)
Imipenem	0│0	0│0	0│0	0│0	0│0	0│0	0│0	0│0	0│0	0│0	0(0%)	0(0%)
Meropenem	0│0	0│0	0│0	0│0	0│0	0│0	0│0	0│0	0│0	0│0	0(0%)	0(0%)
Glycylcyclines
Tigecycline	0│0	0│0	0│0	0│0	0│0	0│0	0│0	0│0	0│0	0│0	0(0%)	0 (0%)
Monobactams
Aztreonam	0│0	0│0	0│0	0│1	0│3	0│0	0│0	0│0	0│0	0│0	0(0%)	4 (10.8%)
Penicillins
Ampicillin	0│1	0│1	0│0	0│1	0│3	0│0	0│3	0│0	0│1	0│9	0(0%)	19(51.4%)
Piperacillin	0│1	0│1	0│0	0│1	0│3	0│0	0│3	0│0	0│1	0│9	0(0%)	19(51.4%)
Ticarcillin	0│1	0│1	0│0	0│1	0│3	0│0	0│3	0│0	0│1	0│9	0(0%)	19(51.4%)
Highly important
Amphenicols
Chloramphenicol	0│1	0│1	0│0	0│1	0│3	0│0	0│3	0│0	0│1	1│6	1(2.7%)	16(43.2%)
Cephalosporins (1st and 2nd gen.)
Cefalotin	0│0	0│0	0│0	0│1	0│3	0│0	0│0	0│0	0│0	0│0	0(0%)	4(10.8%)
Cefoxitin	0│0	0│0	0│0	0│0	3│0	0│0	0│0	0│0	0│0	0│0	3(8.1%)	0(0%)
Cefuroxime	0│0	0│0	0│0	0│1	0│3	0│0	0│0	0│0	0│0	2│0	2(5.4%)	4(10.8%)
Cefuroxime Axetil	0│0	0│0	0│0	0│1	0│3	0│0	0│0	0│0	0│0	2│0	2(5.4%)	4(10.8%)
Tetracyclines
Minocycline	0│0	0│0	0│0	0│1	0│3	0│0	0│3	0│0	0│1	0│9	0(0%)	17(45.9%)
Tetracycline	0│1	0│0	0│0	0│1	0│3	0│0	0│3	0│0	0│1	0│9	0(0%)	18(48.6%)
Other
Penicillin & β-lactamase inhibitor
Amoxicillin/Clavulanic Acid	1│0	0│0	0│0	0│0	0│0	0│0	0│0	0│0	0│0	0│0	1(2.7%)	0(0%)
Piperacillin/Tazobactam	0│0	0│0	0│0	0│0	0│0	0│0	0│0	0│0	0│0	0│0	0(0%)	0(0%)
Folate pathway inhibitors
Trimethoprim	0│1	0│0	0│0	0│3	0│0	0│0	0│3	0│0	0│0	0│9	0(0%)	16(43.2%)
Folate pathway inhibitors/Sulfonamides
Trimethoprim/Sulfamethoxazole	0│1	0│0	0│0	0│3	0│0	0│0	0│3	0│0	0│1	0│9	0(0%)	17(45.9%)
Nitrofurantoins
Nitrofurantoin	0│0	0│0	0│0	6│1	0│3	0│0	0│0	0│0	0│0	0│0	6(16.2%)	4(10.8%)
ESBL (%)	–	–	–	1 (7.7%)	3 (100%)	–	–	–	–	–	–	4(10.8%)
MDR (%)	1(100%)	1 (100%)	–	1(7.7%)	3(100%)	–	3(100%)	–	1(100%)	9(100%)	–	19(51.4%)

Table C.3

Class and antimicrobial	Group E (n = 21)
	Anatum	Give	London	Senftenberg	Weltevreden	Intermediate resistant (%)	Fully resistant (%)
No. isolates	11	4	1	3	2
Samples (n = 30) (%)	5 (16.7%)	2 (6.7%)	1 (3.3%)	1 (3.3%)	1 (3.3%)
Highest priority-critically important
Cephalosporins (3rd & 4th gen.)
Cefepime	0│0	0│0	0│0	0│0	0│0	0(0%)	0 (0%)
Cefixime	0│0	0│3	0│0	0│0	0│0	0(0%)	3(14.3%)
Cefotaxime	0│0	0│3	0│0	0│0	0│0	0(0%)	3(14.3%)
Ceftazidime	0│0	0│3	0│0	0│0	0│0	0(0%)	3(14.3%)
Ceftriazone	0│0	0│3	0│0	0│0	0│0	0(0%)	3(14.3%)
Polyymyxins
Colistin	0│0	0│0	0│0	0│0	0│0	0 (0%)	0 (0%)
Quinolones
Ciprofloxacin	2│0	0│4	0│0	0│0	0│0	2(9.5%)	4 (19%)
Levofloxacin	0│0	0│4	0│0	0│0	0│0	0(0%)	4 (19%)
Moxifloxacin	0│0	0│4	0│0	0│0	0│0	0(0%)	4 (19%)
Nalidixic Acid	0│0	0│4	0│0	0│0	0│0	0(0%)	4 (19%)
Ofloxacin	0│0	0│4	0│0	0│0	0│0	0(0%)	4 (19%)
High priority-critically important
Aminoglycosides
Amikacin	0│0	0│0	0│0	0│0	0│0	0(0%)	0 (0%)
Gentamicin	0│0	0│3	0│0	0│0	0│0	0(0%)	3(14.3%)
Tobramycin	0│0	0│3	0│0	0│0	0│0	0(0%)	3(14.3%)
Carbapenems and other penems
Ertapenem	0│0	0│0	0│0	0│0	0│0	0(0%)	0(0%)
Imipenem	0│0	0│0	0│0	0│0	0│0	0(0%)	0(0%)
Meropenem	0│0	0│0	0│0	0│0	0│0	0(0%)	0(0%)
Glycylcyclines
Tigecycline	0│0	0│3	0│0	0│0	0│0	0(0%)	3(14.3%)
Monobactams
Aztreonam	0│0	0│3	0│0	0│0	0│0	0(0%)	3(14.3%)
Penicillins
Ampicillin	0│7	0│4	0│1	0│0	0│0	0(0%)	12(57.1%)
Piperacillin	0│7	0│4	0│1	0│0	0│0	0(0%)	12(57.1%)
Ticarcillin	0│7	0│4	0│1	0│0	0│0	0(0%)	12(57.1%)
Highly important
Amphenicols
Chloramphenicol	0│3	0│4	0│1	0│0	0│0	0(0%)	8(38.1%)
Cephalosporins (1st and 2nd gen.)
Cefalotin	0│0	0│3	0│0	0│0	0│0	0 (0%)	3(14.3%)
Cefoxitin	0│0	0│0	0│0	0│0	0│0	0 (0%)	0(0%)
Cefuroxime	5│0	0│3	0│0	0│0	0│0	5(23.8%)	3(14.3%)
Cefuroxime Axetil	11│0	0│3	0│0	0│0	0│0	11(52.4%)	3(14.3%)
Tetracyclines
Minocycline	3│3	0│4	0│1	0│0	0│0	3(14.3%)	8(38.1%)
Tetracycline	0│10	0│4	0│1	0│0	0│0	0(0%)	15(71.4%)
Other
Penicillin & β-lactamase inhibitor
Amoxicillin/Clavulanic Acid	0│0	0│0	0│0	0│0	0│0	0(0%)	0(0%)
Piperacillin/Tazobactam	0│0	0│0	0│0	0│0	0│0	0(0%)	0(0%)
Folate pathway inhibitors
Trimethoprim	0│9	0│4	0│1	0│0	0│0	0(0%)	14(66.7%)
Folate pathway inhibitors/Sulfonamides
Trimethoprim/Sulfamethoxazole	0│3	0│4	0│1	0│0	0│0	0(0%)	8(38.1%)
Nitrofurantoins
Nitrofurantoin	0│0	0│0	0│0	0│0	0│0	0(0%)	0 (0%)
ESBL (%)		3 (75%)	–	–	–	–	3(14.3%)
MDR (%)	10(90.9%)	4(100%)	1(100%)	–	–	–	15(71.4%)

Table C.4

Class and antimicrobial	Other serogroups (N = 11)
	Enteritidis	Kedougou	Poona	Urbana	Intermediate resistant (%)	Fully resistant (%)
No. isolates	4	2	3	2
Samples (n = 30) (%)	2 (6.7%)	1 (3.3%)	1 (3.3%)	1 (3.3%)
Highest priority-critically important
Cephalosporins (3rd & 4th gen.)
Cefepime	0│0	0│0	0│0	0│0	0(0%)	0(0%)
Cefixime	0│0	0│0	0│0	0│0	0(0%)	0(0%)
Cefotaxime	0│0	0│0	0│0	0│0	0(0%)	0(0%)
Ceftazidime	0│0	0│0	0│0	0│0	0(0%)	0(0%)
Ceftriazone	0│0	0│0	0│0	0│0	0(0%)	0(0%)
Polyymyxins
Colistin	0│1	0│0	0│0	0│0	0(0%)	1(9.1%)
Quinolones
Ciprofloxacin	3│0	1│0	0│0	0│0	4(36.4%)	0 (0%)
Levofloxacin	3│0	1│0	0│0	0│0	4(36.4%)	0 (0%)
Moxifloxacin	0│0	0│0	0│0	0│0	0 (0%)	0 (0%)
Nalidixic Acid	0│3	0│0	0│0	0│0	0 (0%)	3 (27.3%)
Ofloxacin	3│0	0│1	0│0	0│0	3 (27.3%)	1 (9.1%)
High priority-critically important
Aminoglycosides
Amikacin	0│0	0│0	0│0	0│0	0(0%)	0(0%)
Gentamicin	0│0	0│0	0│0	0│0	0(0%)	0(0%)
Tobramycin	0│0	0│0	0│0	0│0	0(0%)	0(0%)
Carbapenems and other penems
Ertapenem	0│0	0│0	0│0	0│0	0(0%)	0(0%)
Imipenem	0│0	0│0	0│0	0│0	0(0%)	0(0%)
Meropenem	0│0	0│0	0│0	0│0	0(0%)	0(0%)
Glycylcyclines
Tigecycline	0│0	0│0	0│0	0│0	0 (0%)	0(0%)
Monobactams
Aztreonam	0│0	0│0	0│0	0│0	0 (0%)	0(0%)
Penicillins
Ampicillin	0│3	0│0	0│0	0│0	0(0%)	3(27.3%)
Piperacillin	0│3	0│0	0│0	0│0	0(0%)	3(27.3%)
Ticarcillin	0│3	0│0	0│0	0│0	0(0%)	3(27.3%)
Highly important
Amphenicols
Chloramphenicol	0│0	0│0	0│0	0│0	0(0%)	0 (0%)
Cephalosporins (1st and 2nd gen.)
Cefalotin	2│0	0│0	0│0	0│0	2(18.2%)	0(0%)
Cefoxitin	0│0	0│0	0│0	0│0	0(0%)	0(0%)
Cefuroxime	0│0	1│0	0│0	0│0	1 (9.1%)	0(0%)
Cefuroxime Axetil	2│0	1│0	0│0	0│0	3(27.3%)	0(0%)
Tetracyclines
Minocycline	0│3	1│0	0│0	0│0	1(9.1%)	3(27.3%)
Tetracycline	0│3	0│0	0│0	0│0	0(0%)	3(27.3%)
Other
Penicillin & β-lactamase inhibitor
Amoxicillin/Clavulanic Acid	1│0	0│0	0│0	0│0	1(9.1%)	0(0%)
Piperacillin/Tazobactam	0│0	0│0	0│0	0│0	0(0%)	0(0%)
Folate pathway inhibitors
Trimethoprim	0│0	0│0	0│0	0│0	0(0%)	0(0%)
Folate pathway inhibitors/Sulfonamides
Trimethoprim/Sulfamethoxazole	0│0	0│0	0│0	0│0	0(0%)	0(0%)
Nitrofurantoins
Nitrofurantoin	0│3	0│0	0│0	0│0	0(0%)	3(27.3%)
ESBL (%)	–	–	–	–	–	0(0%)
MDR (%)	3(75%)	–	–	–	–	3(27.3%)

Table C.5

Class and antimicrobial Isolates (n = 90)	All serovars			CLSI 2017 breakpoints
	No. isolates (N = 90)	Intermediate resistant (%)	Fully resistant (%)
				Sensitive	Resistant
Highest priority-critically important
Cephalosporins (3rd & 4th gen.)
Cefepime	0│0	0(0%)	0(0%)	≤2	≥16
Cefixime	0│7	0(0%)	7(7.8%)	≤1	≥4
Cefotaxime	0│7	0(0%)	7(7.8%)	≤1	≥4
Ceftazidime	0│3	0(0%)	3(3.3%)	≤4	≥16
Ceftriazone	0│7	0(0%)	7(7.8%)	≤1	≥4
Polyymyxins
Colistin	0│1	0 (0%)	1(1.1%)	≤2	>2
Quinolones
Ciprofloxacin	23│30	23(25.6%)	30(33.3%)	≤0.06	≥1
Levofloxacin	33│14	33(36.7%)	14(15.6%)	≤0.12	≥2
Moxifloxacin	3│13	3(3.3%)	13(14.4%)	≤2	≥8
Nalidixic Acid	0│22	0(0%)	22(24.4%)	≤16	≥32
Ofloxacin	4│43	4(4.4%)	43(47.8%)	≤0.12	≥2
High priority-critically important
Aminoglycosides
Amikacin	0│0	0(0%)	0(0%)	≤16	≥64
Gentamicin	0│15	0(0%)	15(16.7%)	≤4	≥16
Tobramycin	10│7	10(11.1%)	7(7.8%)	≤4	≥16
Carbapenems and other penems
Ertapenem	0│0	0(0%)	0(0%)	≤0.5	≥2
Imipenem	0│0	0(0%)	0(0%)	≤1	≥4
Meropenem	0│0	0(0%)	0(0%)	≤1	≥4
Glycylcyclines
Tigecycline	3│3	3(3.3%)	3(3.3%)	≤2	≥8
Monobactams
Aztreonam	0│7	0(0%)	7(7.8%)	≤4	≥16
Penicillins
Ampicillin	0│50	0(0%)	50(55.6%)	≤8	≥32
Piperacillin	0│50	0(0%)	50(55.6%)	≤16	≥128
Ticarcillin	1│49	1(1.1%)	49(54.4%)	≤16	≥128
Highly important
Amphenicols
Chloramphenicol	1│38	1 (1.1%)	38 (42.2%)	≤8	≥32
Cephalosporins (1st and 2nd gen.)
Cefalotin	3│7	3(3.3%)	7(7.8%)	≤8	≥32
Cefoxitin	3│0	3(3.3%)	0 (0%)	≤8	≥32
Cefuroxime	11│7	11(12.2%)	7(7.8%)	≤8	≥32
Cefuroxime Axetil	19│7	19(21.1%)	7(7.8%)	≤4	≥32
Tetracyclines
Minocycline	7│44	7(7.8%)	44(48.9%)	≤4	≥16
Tetracycline	1│51	1(1.1%)	51(56.7%)	≤4	≥16
Other
Penicillin & β-lactamase inhibitor
Amoxicillin/Clavulanic Acid	3│0	3(3.3%)	0(0%)	≤8/4	≥32/16
Piperacillin/Tazobactam	0│0	0(0%)	0(0%)	≤16/4	≥128/4
Folate pathway inhibitors
Trimethoprim	0│41	0(0%)	41(45.6%)	≤8	≥16
Folate pathway inhibitors/Sulfonamides
Trimethoprim/Sulfamethoxazole	0│38	0(0%)	38(42.2%)	≤2/38	≥4/76
Nitrofurantoins
Nitrofurantoin	6│7	6(6.7%)	7 (7.8%)	≤32	≥128
ESBL (%)	–	–	7(7.8%)	–	–
MDR (%)	–	–	53(58.9%)	–	–

Table D

Phenotypic antimicrobial susceptibility results for Vibrio spp. isolates (n = 133). The figures correspond to the number of intermediate resistant, followed by the number of fully resistant isolates.

Class and antimicrobial	No. isolates and percent resistant (%) (n = 133 isolates)								Disk	Zone diameter (mm)
	V. parahaemolyticus (n = 64)	V. navarrensis (n = 23)	V. alginolyticus (n = 21)	V. cholerae non-O1 (n = 15)	V. vulnificus (n = 6)	V. fluvialis (n = 4)	Total intermediate resistant (%)	Total fully resistant (%)	antimicrobial
									(μg/mL)	Sensitive	Resistant
Highest priority-critically important
Cephalosporins (3rd &4th gen.)
Cefotaxime	3ǀ20	10ǀ2	0ǀ0	8ǀ0	2ǀ0	0ǀ0	23 (17.3%)	22 (16.5%)	30	≥23	≤14
Ceftazidime	3ǀ10	0ǀ0	0ǀ0	1ǀ1	0ǀ0	0ǀ0	4 (3.0%)	11 (8.3%)	30	≥18	≤14
Quinolones
Ciprofloxacin	9ǀ7	0ǀ0	1ǀ0	2ǀ0	1ǀ0	0ǀ0	13 (9.8%)	7 (5.3%)	5	≥21	≤15
Ofloxacin	3ǀ6	0ǀ0	0ǀ0	2ǀ0	0ǀ0	0ǀ0	5 (3.8%)	6 (4.5%)	5	≥16	≤12
High priority-critically important
Aminoglycosides
Amikacin	29ǀ1	1ǀ0	1ǀ0	0ǀ0	1ǀ0	0ǀ0	32 (24.0%)	1 (0.8%)	30	≥17	≤14
Gentamicin	7ǀ0	1ǀ0	1ǀ0	0ǀ0	0ǀ0	0ǀ0	9 (6.8%)	0 (0%)	10	≥15	≤12
Carbapenems and other penems
Imipenem	0ǀ2	0ǀ1	0ǀ0	1ǀ0	0ǀ0	0ǀ0	1 (0.8%)	3 (2.3%)	10	≥16	≤13
Penicillins
Ampicillin	0ǀ64	6ǀ12	0ǀ21	3ǀ8	1ǀ2	0ǀ3	10 (7.5%)	110 (82.7%)	10	≥17	≤13
Amoxicillin-clavulanic acid	1ǀ6	8ǀ4	0ǀ0	3ǀ5	2ǀ0	3ǀ0	17 (12.8%)	15 (11.3%)	20ǀ10	≥18	≤13
Highly important
Amphenicols
Chloramphenicol	3ǀ1	1ǀ0	0ǀ0	1ǀ0	1ǀ0	0ǀ0	6 (4.5%)	1 (0.8%)	30	≥18	≤12
Tetracyclines
Tetracycline	15ǀ7	3ǀ1	0ǀ0	1ǀ1	0ǀ1	0ǀ0	19 (14.3%)	10 (7.5%)	30	≥19	≤14
Folate pathway inhibitors & sulfonamides
Trimethoprim/sulfamethoxazole	7ǀ17	3ǀ3	1ǀ0	0ǀ3	1ǀ2	1ǀ0	13 (9.8%)	25 (18.8%)	1.25/23.75	≥16	≤10
ESBL	18	0	0	0	0	0	–	18 (13.5%)
MDR	18	0	0	0	0	0	–	18 (13.5%)