Occurrence of blaNDM-1-Positive Providencia spp. in a Pig Farm of China

Antibiotics have been extensively used to ensure the productivity of animals on intensive livestock farms. Accordingly, antimicrobial-resistant organisms, which can be transmitted to humans via the food chain, pose a threat to public health. The Enterobacterium antimicrobial resistance gene, blaNDM-1, is a transmissible gene that has attracted widespread attention. Here, we aimed to investigate the prevalence of Enterobacteriaceae carrying blaNDM-1 on an intensive pig farm. A total of 190 samples were collected from a pig farm in Hunan Province, China. Resistant isolates were selected using MacConkey agar with meropenem and PCR to screen for blaNDM-1-positive isolates. Positive strains were tested for conjugation, antimicrobial susceptibility, and whole-genome sequencing. Four blaNDM-1-positive Providencia strains were obtained, and multidrug resistance was observed in these strains. The structure carrying blaNDM-1 did not conjugate to E. coli J53 after three repeated conjugation assays. This suggests that, in intensive farming, attention should be focused on animal health and welfare to reduce the frequency of antibiotic usage. Carbapenem-resistant Enterobacteriaceae in the breeding industry should be included in systematic monitoring programs, including animal, human, and environmental monitoring programs.


Introduction
Antimicrobial resistance (AMR) is recognized as one of the most serious threats to human and animal health [1][2][3]. These concerns are amplified by the rapid increase in carbapenem-resistant Enterobacteriaceae (CRE), which can carry and spread resistance genes [4]. Infections by CRE are associated with significant morbidity and mortality [5], as well as substantial economic loss.
The mechanisms underlying carbapenem resistance in CRE are complex and are known to involve the production of the enzyme New Delhi metallo-beta-lactamase (NDM-1), a carbapenemase encoded by the bla NDM-1 gene [6]. Since the first report of a strain of bla NDM-1positive Klebsiella pneumoniae from India in 2008 [7], many Enterobacteriaceae containing bla NDM-1 have been reported worldwide [8][9][10]. Several bla NDM-1 -positive isolates belong to the Enterobacteriaceae genus Providencia, which consists of several gram-negative opportunistic pathogenic strains [11]. Providencia spp. have been reported to cause diarrhea in

Identification of bla NDM-1 -Positive Isolates
Ten strains were screened using meropenem (0.5 µg/mL): three strains were obtained from environmental samples from three different rooms (breeding rooms, pregnancy rooms, and delivery rooms), and six strains were obtained from fecal samples (three from the piglet rooms and three from fattening rooms). PCR analysis of these 10 samples revealed four bla NDM-1 -positive bacterial strains. Three of the four bla NDM-1 -positive strains (20Q122mw, 20Q124mw, and 20Q126mw) were obtained from pig fecal samples collected from the piglet room. The other bla NDM-1 -positive strain (20Q171mw) was obtained from a pig fecal sample collected from a fattening room. Bacterial 16S rRNA sequencing identified strains 20Q122mw and 20Q124mw as Providencia rettgeri and strains 20Q126mw and 20Q171mw as P. stuartii.

Antimicrobial Susceptibility Profiles and Conjugation Experiments
The multidrug resistance of the four bla NDM-1 -positive Providencia strains was assessed by performing drug sensitivity tests (Table 1). Multidrug-resistant bacteria were defined as those resistant to three or more classes of antibiotics [26]. The minimum inhibitory concentrations (MICs) of florfenicol and colistin against the bla NDM-1 -positive strains exceeded 128 µg/mL, which indicated high resistance. Resistance to florfenicol can be attributed to the presence of the amphenicol resistance gene, floR, in both P. rettgeri and P. stuartii (Table 2). Meanwhile, resistance to colistin likely corresponds to the natural antimicrobial resistance of Providencia since no resistance genes against colistin have been previously found [27]. Although all four bla NDM-1 -positive strains exhibited multidrug resistance, some differences in the resistance profiles between the P. rettgeri and P. stuartii strains were observed. In particular, a difference was observed in the susceptibility to gentamicin, wherein the P. stuartii strains were resistant to gentamicin up to a concentration of 16 µg/mL, whereas the P. stuartii strains were sensitive to 0.25 µg/mL gentamicin.
No strains with successful conjugation were obtained after three repeat tests.

Discussion
To the best of our knowledge, this study is the first to identify bla NDM-1 in Providencia spp. isolated from pigs. These strains were obtained from pig fecal samples from a pig farm in China, specifically from piglet and fattening rooms. As members of the Enterobacteriaceae family, the discovery of bla NDM-1 -positive Providencia in food-producing animals is of concern. Enterobacteriaceae harboring bla NDM-1 are resistant to many antibacterial agents and cause problems in the treatment of many bacterial infections in animals and humans. Genetic segments containing bla NDM-1 can conjugate with different bacteria via mobile genetic elements, leading to the spread of multidrug resistance [29][30][31]. Although the four bla NDM-1 -positive Providencia strains identified in this study had inserted sequences around bla NDM-1 , they did not conjugate with E. coli J53 after three repeated conjugation assays. This might be due to preferential conjugation effects between bacteria; for example, the insert sequences of the IS30 family were observed to preferentially insert into the inverted repeat sequence of similar regions than with themselves [32]. It was possible that the conditions of our conjugation tests did not meet the conditions for transfer or that transfer to E. coli J53 was not compatible with the bla NDM-1 -positive Providencia strains.
Among the various bacterial genera present in the samples collected in this study, bla NDM-1 was only detected in Providencia strains. Therefore, Providencia strains were probably the dominant population for bla NDM-1 transmission in this pig farm. These isolates were collected from different rooms (piglet and fattening rooms) and identified as different species (P. rettgeri and P. stuartii); however, all isolates harbored the same bla NDM-1 segment. This suggests that horizontal gene transfer is a probable route of transmission in this farm. Furthermore, whole genome sequencing analyses identified consistent clusters of highly genetically-related isolates (20Q122mw and 20Q124mw; 20Q126mw and 20Q171mw), suggesting the possibility of bla NDM-1 vertical transmission.
On intensive pig farms, personnel disinfection is usually strictly enforced to ensure the health of animals, and meropenem use is not allowed. However, bla NDM-1 -positive Providencia strains were detected. Thus, it is possible that the presence of bla NDM-1 on this pig farm might have originated from Providencia harboring bla NDM-1 in wild hosts, such as wild birds, flies, and mice. Preventing exposure to wild host species may help prevent the transfer of bla NDM-1 -carrying strains to pigs.
In addition, we noted some interesting observations while analyzing the genetic environment near bla NDM-1 . Compared to the pNDM-PM58 plasmid of P. mirabilis isolated from a human sample, a similar segment (QacE-sul1-IS91-trpF-ble-bla NDM-1 -ISAba125) was found in the bla NDM-1 -positive Providencia strains. While a similar segment shared by different bacteria is not necessarily proof of horizontal gene transfer as they might originate from different sources [33], this segment that contains bla NDM-1 has the potential to spread between humans and animals.

Materials and Methods
In December 2020, 190 fecal (n = 137) or environmental (n = 53) samples were collected from a pig farm in Hunan Province, China. Pig fecal samples were collected from the following areas: breeding rooms (n = 28), pregnancy rooms (n = 27), delivery rooms (n = 44), piglet rooms (n = 26), and fattening rooms (n = 12). Environmental samples, including from pig drinking water, sewage, and dirt, were collected from the following areas: breeding rooms (n = 9), pregnancy rooms (n = 11), delivery rooms (n = 18), piglet rooms (n = 9), and fattening rooms (n = 6). All samples were collected using sterile swabs and were then suspended in 1 mL phosphate-buffered saline. These samples were stored in iceboxes and transported to the laboratory at the end of sampling. MacConkey agar (Landbridge, Beijing, China) supplemented with 0.5 µg/mL meropenem (Meilun Biotechnology Co., Ltd., Dalian, China) and vancomycin (30 µg/mL, Meilun Biotechnology Co. Ltd., Dalian, China) was used to screen for antibiotic-resistant bacterial strains in the samples. PCR and electrophoresis assays were performed to verify whether the filtered isolates contained bla NDM-1 [34]. The 16S rRNA sequencing was performed to confirm the presence of the bacterial species by Tsingke Biotechnology Company (Changsha, China).
Subsequently, bla NDM-1 -positive strains were used as donor bacteria and sodium azidetolerant E. coli J53 as receptor bacteria. The donor and receptor bacteria were mixed at a ratio of 1:3 in LB broth (Landbridge, Beijing, China), and the mixture was spread onto microporous membranes affixed to Mueller Hinton agar (Landbridge, Beijing, China) at 37 • C for 18 h. The colonies on the membranes were then diluted in LB broth and evenly coated onto MacConkey agar plates containing 1 µg/mL meropenem and 400 µg/mL sodium azide. The cultures were incubated at 37 • C for 18 h. Then, the strains were filtered using these cultures. Conjugation assays were performed in triplicate.
DNA from all bla NDM-1 -positive isolates was extracted using the TIANamp Bacteria DNA Kit (Tiangen Biotech Co., China), according to the manufacturer's instructions. WGS of 20Q122mw, 20Q124mw, and 20Q171mw was performed using the Illumina HiSeq sequencing platform (Illumina, San Diego, CA, USA) by Annoroad Gene Technology (Beijing, China). Meanwhile, genomic DNA sequencing of strain 20Q126mw was performed using the Nanopore MinION (100-fold average read depth). Raw sequences were assembled using SPAdes 3.11 (Bankevich et al., 2012) and annotated using RAST (http://rast.nmpdr. org/, accessed on 8 January 2022). AMR genes were searched in the Center for Genomic Epidemiology database (www.genomicepidemiology.org, accessed on 8 January 2022). The Basic Local Alignment Search Tool (BLAST) of the National Center for Biotechnology Information (NCBI) (https://blast.ncbi.nlm.nih.gov/Blast.cgi, accessed on 16 January 2022) was used to analyze the alignments of similar sequences. The genetic environment of bla NDM-1 was investigated using Easyfig 2.2.5 [35]. The BLAST Ring Image Generator (BRIG 0.9516) was used to generate a comparative genomic circle map [36].

Conclusions
In this study, four strains of bla NDM-1 -positive Providencia spp. were found in the feces of swine. These four strains carried the same segments of bla NDM-1 and were isolated from different farming rooms. Furthermore, the segments were similar to that of Proteus mirabilis, which was isolated from a patient in Zhengzhou, China, thereby indicating that these segments of bla NDM-1 can be transmitted between humans and animals. In order to reduce the risk of AMR transmission, the conditions in pig farms should be further optimized to minimize the use of antibiotics. AMR monitoring of intensive farms should be maintained to keep humans and animals safe.