Listeria monocytogenes antibiotic resistance

Listeria monocytogenes is an important foodborne pathogen worldwide, that can cause life-threatening listeriosis disease in vulnerable groups including pregnant women, fetuses, elderly people, and immunocompromised individuals, with a considerable mortality rate [20–30%] [Lomonaco et al., 2009]. L. monocytogenes is capable of proliferating in different stressful environmental conditions, including high salinity, low temperature, and a wide range of pH values. Foodborne listeriosis poses a global economic and health burden due to the wide spread of L. monocytogenes in food and food processing environments.

In China, 147 clinical cases in total and 82 outbreak-related cases were reported in 28 provinces from January 1964 to December 2010 [Feng et al., 2013]. In recent years, listeriosis diseases have been increasingly reported in China annually, especially in developed cities [Wang et al., 2015, 2018]. This trend may be linked to the trend of direct consumption of fresh foods and ready-to-eat foods in China, especially in developed cities. However, there is no national monitoring system for listeriosis cases in China as it is not yet considered a notifiable disease. According to the report of the European Food Safety Authority [EFSA], the small number of listeriosis outbreaks may be linked to the lower consumption of fish and fish products compared to other foods in Europe [European Food Safety Authority [EFSA] and European Centre for Disease Control [ECDC], 2015]. Similarly, as reported by Song et al. [2015], ready-to-eat [RTE] raw fish has the highest per serving risk of listeriosis among the five kinds of ready-to-eat foods based on risk assessment. This indicates that RTE fish and fish products may serve as a source of L. monocytogenes infection. Previous studies have focused on the prevalence of L. monocytogenes in aquatic products, but these studies only focused their investigation to limited cities/provinces, such as Jiangsu Province, Henan Province, Beijing City, and Zhanjiang City [Chen et al., 2013; Ma, 2015; Wu et al., 2017; Chui et al., 2018]. However, little information is available on the comprehensive investigation and potential virulence of L. monocytogenes from aquatic products in China. According to data compiled by the National Bureau of Statistics of China, the average consumption of aquatic products per capita has increased annually and in 2016, it was 11.4 kg . In this context, it is necessary to systematically investigate the contamination level of L. monocytogenes in aquatic products in China.

Listeria monocytogenes has been differentiated into 13 serotypes based on its somatic [O] and flagellar [H] antigens, and are further grouped into five serovars, designated as serogroups I.1 [1/2a-3a], I.2 [1/2c-3c], II.1 [4b-4d-4e], II.2 [1/2b-3b-7], and III [4a-4c] [Doumith et al., 2004]. A series of virulence factors participating in cellular infection cycles have been documented in L. monocytogenes. Listeria pathogenicity island-1 [LIPI-1, including prfA, hly, mpl, iap, plcB, plcC, and actA] and LIPI-2 [inlA and inlB] are considered as the two classic major pathogenicity islands in L. monocytogenes. L. monocytogenes is known to exhibit varied pathogenicity in intra-species isolates even though each isolate harbors both LIPI-1 and LIPI-2. In recent years, the llsX gene [encoding Listeriolysin, LLS, a hemolytic, and cytotoxic factor] belonging to LIPI-3, has been greatly associated with a subset of lineage I in human listeriosis [Cotter et al., 2008]. Maury et al. [2016] identified that a cluster of six genes annotated as the cellobiose-family phosphotransferase system [PTS] and designated as LIPI-4, was highly associated with L. monocytogenes neuroinvasiveness and human maternal-neonatal [MN] infection. At present, LIPI-3 were found only in lineage I clonal complexes, 23 STs were found to carried LIPI-3 [ST1, ST1001, ST3, ST4, ST6, ST191, ST213, ST217, ST224, ST288, ST363, ST79, ST77, ST382, ST389, ST489, ST999, ST554, ST581, ST1000, ST380, ST778, and ST619], LIPI-4 presented in 11 STs belongs to lineage I and a single lineage III [ST4, ST87, ST213, ST217, ST363, ST382, ST388, ST663, ST1002, ST1166, and ST619] [Chen et al., 2018; Kim et al., 2018; Wang et al., 2018]. The presence of these virulence genes may be an important determinant to their pathogenicity.

The objective of this study was to [i] comprehensively investigate the occurrence and contamination level of L. monocytogenes in fresh aquatic products; [ii] evaluate the potential virulence and antibacterial profiles of L. monocytogenes isolates; and [iii] explore the genetic diversity of L. monocytogenes isolates recovered from the Chinese retail aquatic system.

Materials and Methods

Samples

From July 2012 to April 2016, a total of 846 retail aquatic products were collected from rural markets [n = 245], open-air markets [n = 261], and large supermarkets [n = 340] from 43 cities of China. The marine aquatic products [n = 506] included squid [n = 150 samples], shrimp [n = 145], yellow croak [n = 100], weever [n = 24], shell fish [n = 23], saury [n = 20], octopus [n = 10], cuttlefish [n = 6], silvery pomfret [n = 3], salmon [n = 6], capelin [n = 3], and other species [n = 16]. Freshwater aquatic products [n = 341] comprised the crucian carp [n = 117], grass carp [n = 82], Tilapia mossambica [n = 49], Megalobrama amblycephala [n = 29], Cyprinus carpio [n = 21], bighead carp [n = 7], Pelteobagrus fulvidraco [n = 5], silver carp [n = 5], Channa argus [n = 4], catfish [n = 2], and other species [n = 20]. Except of packaged salmon products, all of marine aquatic products except shrimp were stored loosely in ice, while freshwater aquatic products were alive keeping in water. Among these samples, salmon, grass carp, and Tilapia mossambica were intended to be eaten raw. All samples were placed in insulated shipping coolers containing frozen gel packs, which were placed on the sides, middle, and the top of the samples. All samples were kept below 4°C during transportation and testing was initiated within 4 h after receiving the samples.

Qualitative and Quantitative Analysis

Qualitative detection of L. monocytogenes was performed according to the National Food Safety Standard of China [4789.30-2010] with minor adaptations Ministry of Health of People’s Republic of China [2010]. Briefly, 25 g of homogenized aquatic samples were added to 225 mL Listeria enrichment broth 1 [LB1] [Guangdong Huankai Co., Ltd., Guangzhou, China]. The cultures in LB1 media were incubated at 30°C for 24 h, after which 0.1 mL LB1 enrichment culture was transferred to 10 mL Listeria enrichment broth 2 [LB2] at 30°C for 24 h. A loopful [about 10 μL] of the LB2 enrichment culture was streaked onto Listeria selective agar plates [Guangdong Huankai Co., Ltd.] and incubated at 37°C for 48 h. Three to five presumptive colonies were selected for the identification of L. monocytogenes using the Microgen ID Listeria identification system [Microgen, Camberley, United Kingdom] according to the manufacturer’s instructions.

The workflow for most probable number [MPN] was adapted according to a previous study by Gombas et al. [2003]. Briefly, a 9-tube MPN method was used. The nine tubes were divided into three sets of three tubes. The first set of tubes was contained 10 mL of the sample homogenate, the second and third sets of tubes contained 10 mL of Fraser broth [Guangdong Huankai Co., Ltd., Guangzhou, China] inoculated with 1 and 0.1 mL of the homogenate, respectively. Three aliquots [10, 1, and 0.1 mL] of the sample homogenate were dispensed into three sets, representing 1.0, 0.1, and 0.01 g of the original sample, respectively. The tubes were incubated at 30 ± 2°C for 24 ± 2 h. Darkened Fraser tubes were subjected to streaking onto Listeria selective agar plates. If a Fraser broth tube did not darken, it was examined again after an additional 26 ± 2 h of incubation. The MPN value was determined based on the number of positive tube[s] in each of the three sets and the MPN table [U.S. Department of Agriculture, 1998].

Serogroup Analysis

Genomic DNA was extracted from L. monocytogenes using a Bacterial Genomic DNA Purification Kit [Magen Biotech Inc., Guangzhou, China] according to the manufacturer’s instructions. Serogroup analysis of 72 isolates was performed using multiplex PCR as described by Doumith et al. [2004] [Supplementary Table S1]. Five distinct serogroups, I.1 [1/2a-3a], I.2 [1/2c-3c], II.1 [4b-4d-4e], II.2 [1/2b-3b-7], and III [4a-4c], were identified using multiplex PCR. The primers used are shown in Supplementary Table S2. PCR was performed with an initial denaturation step at 94°C for 3 min; 35 cycles of 94°C for 35 s, 53°C for 50 s, and 72°C for 60 s; and a final cycle of 72°C for 7 min in a thermocycler [Applied Biosystems, CA, United States]. The amplicons [8 μl] were separated on 2% agarose gels in TAE buffer and then visualized by Goldview® staining [0.005%, v/v]. All strains were serotyped by antigen serum agglutination according to the manufacturer’s instruction [Denka Seiken Co., Ltd., Tokyo, Japan].

Antimicrobial Susceptibility Test

All strains collected were analyzed by the KB method using breakpoints recommended by the National Committee for Clinical Laboratory Standards [Clinical and Laboratory Standards Institute, 2014] for Staphylococcus, except for ampicillin and penicillin G where specific Listeria breakpoints are defined [M45-A2 Vol. 30 No. 18] [Supplementary Table S3]. In total, 16 antibiotic agents, including those used to treat human listeriosis, were tested by the KB method as follows: ampicillin [AMP; 10 μg], chloramphenicol [C; 30 μg], erythromycin [E; 15 μg], gentamicin [CN; 10 μg], kanamycin [K; 30 μg], rifampin [RD; 5 μg], doxycycline [DO, 30 μg], penicillin [P, 10 U], tetracycline [TE; 30 μg], vancomycin [VA; 30 μg], sulfamethoxazole with trimethoprim [SXT; 23.75/1.25 μg], sulbactam/ampicillin [SAM; 10/10 μg], meropenem [MEM; 10 μg], linezolid [LZD, 30 μg], and amoxycillin/clavulanic acid [AMC; 10 μg] [Oxoid, Basingstoke, United Kingdom]. Staphylococcus aureus ATCC 25923 and Escherichia coli ATCC 25922 were used as quality control strains. Zones of inhibition were measured with precision calipers to the nearest 0.01 mm. Isolates exhibiting resistance to at least three classes of the antimicrobial agents tested, were considered multidrug-resistant strains.

Identification of Potential Hypervirulent Isolates

To evaluate the potential virulence of 72 L. monocytogenes isolates, the presence of virulence factors associated with infection cycle and invasiveness was detected by PCR. As shown in Supplementary Table S4, nine virulence factor genes belonging to LIPI-1 and LIPI-2 were detected by PCR. In addition, the presence of LIPI-3 and LIPI-4 genes [llsX and ptsaA, respectively] was also observed by PCR [Clayton et al., 2011; Maury et al., 2016]. The premature stop codons [PMSCs] of inlA were determined by amplicon sequencing. The complete length of inlA [2403 bp] was sequenced in 72 isolates. External primers were used to amplify the complete inlA gene and internal primers were used for sequencing [Supplementary Table S3; Wu et al., 2016]. The inlA sequences were assembled using DNAMAN software [version 8th]. By comparing the obtained complete inlA sequence to that of the L. monocytogenes EGDe [Glaser et al., 2001], PMSC types were determined according to the site of PMSC-mutation in inlA gene as documented by Gelbicova et al. [2015].

Multilocus Sequence Typing

MLST analysis of L. monocytogenes was previously reported by Ragon et al. [2008] [Supplementary Table S5]. Briefly, each 50 μL PCR contained 5.0 μL 10× PCR buffer [TAKARA, Dalian, China], 1.5 mM MgCl2, 0.2 mM of each dNTP, 0.4 mM of each primer, 1.25 U Taq polymerase, and 1 μL genomic DNA. PCR was performed using the following program: 3 min initial denaturation at 94°C and 35 cycles consisting of denaturation at 94°C for 30 s, annealing at 52°C [45°C for bglA] for 1 min and elongation at 72°C for 2 min, followed by a final elongation for 10 min at 72°C. The PCR products were purified and sequenced by Invitrogen [Thermo Fisher, Shanghai, China]. An allele number was given according to each variant locus of each housekeeping gene; sequence types [STs] and clonal complexes [CCs] were assigned via the Listeria MLST database at the Pasteur Institute website. A neighbor-joining tree of L. monocytogenes based on the MLST of seven housekeeping genes was constructed using MEGA 7.0 [Kumar et al., 2016] with 1000 bootstrap replications. Simpson’s indexes of discrimination [DI] for MLST were calculated to determine the ability of the MLST typing method according to a previous study reported by Hunter and Gaston [1988].

Data Analysis

The statistically significant difference of prevalence of L. monocytogenes between marine aquatic products and freshwater aquatic products was calculated using the Chi-square test. P-values of