*Staphylococcus chromogenes* from bovine sub-clinical mastitis in Sweden

Karin Persson Waller<fnref n="1" />, Mattias Myrenås<fnref n="1" />, Stefan Börjesson<fnref n="2" />, Hyeyoung Kim<fnref n="3" />, Micael Widerström<fnref n="4" />, Tor Monsen<fnref n="4" />, Arnar Sandholt Sigurdarson<fnref n="5" />, Emma Östlund<fnref n="6" />, Wonhee Cha<fnref n="3" />

In Sweden bovine subclinical mastitis is commonly caused by *Staphylococcus chromogenes*. However, knowledge on genetic relatedness, virulence factors and genes encoding antibiotic resistance is overall scarce for *S. chromogenes*. The current study therefore performed genome sequencing and bioinformatic analysis on 105 *S. chromogenes* isolates. The isolates had previously been obtained from Swedish dairy cows with subclinical mastitis in a prior observational study [1]. Isolates were characterized using a 7 locus multi locus sequence typing (7-MLST), core genome analysis, detection of genes encoding AMR, and genetic detection of prospective virulence factors. The isolates belonged to 47 STs, with the most common being ST6 and ST109, and were defined to belong to 7 distinct core genome clusters. Isolates were generally susceptible to antibiotics with only the genes *blaZ* and *str* identified in a limited number of isolates. *S. chromogenes* were on average positive for around 30 prospective virulence factors, with a variation of types and numbers of factors identified in clusters and STs.

**Reference**

[1] Nyman, et.al. (2018). Intramammary infections with different non-aureus staphylococci in dairy cows. *J Dairy Sci* 101:1403-1418.

<footnote n="1">¹Department of Animal Health and Antimicrobial Strategies, National Veterinary Institute (SVA), Uppsala, Sweden</footnote>

<footnote n="2">²School of Health Science, Örebro University, Örebro, Sweden</footnote>

<footnote n="3">³Department of Disease Control and Epidemiology, National Veterinary Institute (SVA), Uppsala Sweden</footnote>

<footnote n="4">⁴Department of Clinical Microbiology, Umeå University, Umeå, Sweden</footnote>

<footnote n="5">⁵deCODE genetics, Reykjavik, Iceland</footnote>

<footnote n="6">⁶Department of Microbiology, National Veterinary Institute (SVA), Uppsala, Sweden</footnote>
# Characterization of an *optrA*-harbouring unconventional circularizable structure located on the novel integrative and conjugative element *ICESsuHN38* in *Streptococcus suis*

Qin YANG<sup>1,4</sup>, Yao ZHU<sup>1</sup>, Stefan SCHWARZ<sup>2,3</sup>, Wanjiang ZHANG<sup>1</sup> and Xiumei WANG<sup>1</sup>

**Objectives:** To identify the novel genetic elements involved in the horizontal transfer of the oxazolidinone/phenicol resistance gene *optrA* [1] in *Streptococcus suis*.

**Methods:** Whole genome DNA of the *optrA*-positive isolate *S. suis* HN38 was subjected to WGS via both Illumina HiSeq and Oxford Nanopore platforms. MICs of several antimicrobial agents were determined by broth microdilution. PCR assays were performed to identify the circular forms of the novel integrative and conjugative element (ICE) *ICESsuHN38*, but also the unconventional circularizable structure (UCS) excised from this ICE. The transferability of *ICESsuHN38* was evaluated by conjugation assays.

**Results:** *S. suis* isolate HN38 harbored the oxazolidinone/phenicol resistance gene *optrA*. The *optrA* gene was flanked by two copies of *erm*(B) genes in the same orientation, located on a novel *ICESa2603* family-like ICE, designated *ICESsuHN38*. PCR assays revealed that a novel UCS [2] carrying the *optrA* and one copy of *erm*(B) could be excised from *ICESsuHN38*. *ICESsuHN38* was able to successfully transfer into the recipient strain *S. suis* BAA by conjugation.

**Conclusions:** In this work, a novel *optrA*-carrying mobile genetic element, an UCS, was identified in *S. suis*. The *optrA* gene was flanked by copies of *erm*(B) and its location on the novel *ICESsuHN38* will aid its horizontal dissemination.

<figure><img src="image_51.png" /><figcaption>**Fig. 1.** Schematic presentation of *ICESsuHN38* and the UCS carrying the *optrA* gene.</figcaption></figure>

**References**

[1] S. Schwarz, W. Zhang, X. D. Du, H. Krüger, A. T. Feßler, et al. (2021). *Clin Microbiol Rev* 3:e0018820.

[2] C. Palmieri, M. Mingoia and P. E. Varaldo (2013). *Antimicrob Agents Chemother* 5:2440-1.

<footnote n="1"><sup>1</sup>State Key Laboratory for Animal Disease Control and Prevention, Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Harbin 150069, China</footnote>

<footnote n="2"><sup>2</sup>Institute of Microbiology and Epizootics, Centre for Infection Medicine, School of Veterinary Medicine, Freie Universität Berlin, 14163 Berlin, Germany</footnote>

<footnote n="3"><sup>3</sup>Veterinary Centre for Resistance Research (TZR), School of Veterinary Medicine, Freie Universität Berlin, 14163 Berlin, Germany</footnote>

<footnote n="4"><sup>4</sup>College of Veterinary Medicine, China Agricultural University, Beijing, China</footnote>
# Identification of ISVlu1-derived translocatable units containing the *optrA* and/or *fexA* genes generated by homologous or illegitimate recombination in *Lactococcus garvieae* of porcine origin

Qin YANG<fnref n="1" />,<fnref n="4" />, Yao ZHU<fnref n="1" />, Stefan SCHWARZ<fnref n="2" />,<fnref n="3" /> and Wanjiang ZHANG<fnref n="1" />

**Objectives:** To investigate the presence and transfer of the oxazolidinone/phenicol resistance gene *optrA* and identify the genetic elements involved in the horizontal transfer of the gene *optrA* [1,2] in *Lactococcus garvieae*.

**Methods:** WGS of the *optrA*-positive isolate *L. garvieae* BN62 was performed using both Illumina HiSeq and Oxford Nanopore platforms. Antimicrobial susceptibility testing by broth microdilution followed EUCAST recommendations. The transferability of plasmid pBN62-*optrA* was evaluated by conjugation assays. Translocatable units (TUs) were detected by PCR assays followed by Sanger sequencing of the amplicons.

**Results:** *L. garvieae* BN62 harbored the oxazolidinone/phenicol resistance gene *optrA*, which along with *fexA* was located on the novel plasmid pBN62-*optrA*. Two complete and three truncated copies of *ISVlu1* elements were found on this plasmid. The *optrA* gene was bounded by two complete *ISVlu1* copies in the same orientation, and the *fexA* gene was bracketed by a complete and a truncated *ISVlu1* in opposite orientations. PCR assays revealed that three different-sized *ISVlu1*-based TUs carrying *optrA* and/or *fexA*, were formed from plasmid pBN62-*optrA*. The TU-*optrA* was generated by homologous recombination while TU-*fexA* and TU-*optrA+fexA* were the products of illegitimate recombinations. Conjugation assays confirmed the successful transfer of plasmid pBN62-*optrA* into *Enterococcus faecalis* JH2-2.

**Conclusions:** To the best of our knowledge, this is first report of *optrA*-harbouring *L. garvieae* of animal origin. Moreover, the formation of three novel *ISVlu1*-mediated TUs underlines that *ISVlu1* is highly active and plays an important role in the transfer of resistance genes.

<figure><img src="image_52.png" /><figcaption>**Fig. 1.** Schematic presentation of three novel TUs, generated from pBN62-optrA.</figcaption></figure>

**References**

[1] S. Schwarz, W. Zhang, X. D. Du, H. Krüger, A. T. Feßler, et al. (2021). *Clin Microbiol Rev* 3:e0018820.

[2] Y. Zhu, W. Yang, S. Schwarz, Q. Xu, Q. Yang, et al. (2022). *J Antimicrob Chemother* 4:921-925.

<footnote n="1">¹State Key Laboratory for Animal Disease Control and Prevention, Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Harbin 150069, China</footnote>

<footnote n="2">²Institute of Microbiology and Epizootics, Centre for Infection Medicine, School of Veterinary Medicine, Freie Universität Berlin, 14163 Berlin, Germany</footnote>

<footnote n="3">³Veterinary Centre for Resistance Research (TZR), School of Veterinary Medicine, Freie Universität Berlin, 14163 Berlin, Germany</footnote>

<footnote n="4">⁴College of Veterinary Medicine, China Agricultural University, Beijing, China</footnote>
Efflux pump's spatial properties control inhibitor binding

Seiji YAMASAKI<fnref n="1" />,<fnref n="2" />,<fnref n="3" />, Naoki KOGA<fnref n="2" />, Martijn ZWAMA<fnref n="3" />, Keisuke SAKURAI<fnref n="3" />, Ryosuke NAKASHIMA<fnref n="3" />, Akihito YAMAGUCHI<fnref n="3" /> and Kunihiko NISHINO<fnref n="2" />,<fnref n="3" />,<fnref n="4" />

Multidrug resistance is facilitated by the multidrug efflux pumps. MexB in *Pseudomonas aeruginosa* and AcrB in *Escherichia coli* are inhibited by the efflux pump inhibitor (EPI) ABI-PP, whereas MexY in *P. aeruginosa* is not. It has already been reported how ABI-PP binds to the inhibitor-binding pits of AcrB and MexB. Bulky tryptophan (Trp) was found to be the cause of the insensitivity of MexY to ABI-PP [1]. Although AcrB (Phe178Trp) is no longer inhibited by ABI-PP and MexY (Trp177Phe) is inhibited by ABI-PP, it is interesting that ABI-PP can inhibit MexB (Phe178Trp). Hence, it is unclear which changes in bulky amino acids are important for inhibitor binding in MexB. Therefore, we further examined the inhibitor binding pit of MexB to determine which Trp mutations inhibit ABI-PP binding. When positions 139, 277, 279, and 612 were changed to tryptophan, the inhibitory effect disappeared. However, the mutation at position 571 had no effect (Fig.1). Our findings suggest that mutations at various sites have a significant impact on the binding effectiveness of EPIs and that spatial factors play a role in EPI binding[2]. Finding novel inhibitors and developing new medications should take these findings into account.

<figure><img src="image_53.png" /><figcaption>Fig. 1. Evaluation of the putative structures and inhibitory effect of ABI-PP in MexB mutants.</figcaption></figure>

**References**

[1] R. Nakashima, K. Sakurai, S. Yamasaki, K. Hayashi, C. Nagata, K. Hoshino, Y. Onodera, K. Nishino and A. Yamaguchi. (2013). *Nature* 500:102-106.

[2] S. Yamasaki, N. Koga, M. Zwama, K. Sakurai, R. Nakashima, A. Yamaguchi and K. Nishino. (2022). *Antimicrob Agents Chemother* 66:e0067222.

<footnote n="1">¹Institute for Advanced Co-Creation Studies, Osaka University, Suita, Japan</footnote>

<footnote n="2">²Graduate School of Pharmaceutical Sciences, Osaka University, Suita, Japan</footnote>

<footnote n="3">³SANKEN, Osaka University, Ibaraki, Japan</footnote>

<footnote n="4">⁴Center for Infectious Disease Education and Research, Osaka University, Suita, Japan</footnote>
# Investigation of the macrolide-resistance in bovine *Mannheimia haemolytica* isolates from Germany

Valeria KOSTOVA<fnref n="1" />,<fnref n="2" />, Henrike KRÜGER-HAKER<fnref n="1" />,<fnref n="2" />, Dennis HANKE<fnref n="1" />,<fnref n="2" />, Heike KASPAR<fnref n="3" />, Stefan SCHWARZ<fnref n="1" />,<fnref n="2" />

*Mannheimia haemolytica* is of considerable importance in the development of the multifactorial bovine respiratory disease (BRD). Recently, data from the German national resistance monitoring program GERM-Vet showed that the number of macrolide-resistant bovine *M. haemolytica* has been slowly increasing since 2009. As bovine respiratory tract infections are often treated with macrolides, this trend may result into severely limited therapeutic options for the management of BRD. In this study, 19 macrolide-resistant bovine *M. haemolytica* isolates from GERM-Vet 2008 – 2020 were investigated. Antimicrobial susceptibility testing (AST) was performed via broth microdilution according to CLSI standards (i) to confirm the macrolide resistance and (ii) to define the resistance phenotypes of the isolates. Minimal inhibitory concentration (MIC) values were obtained for the macrolides erythromycin, tilmicosin, tulathromycin, gamithromycin, and tildipirosin as well as for 22 other antimicrobial agents. All isolates either had elevated MICs or were resistant to at least one or more of the macrolides tested. In particular, isolates Mh190176, Mh191452, and Mh192916 showed resistance to tilmicosin, tulathromycin, gamithromycin, and tildipirosin, and elevated MICs for erythromycin. Furthermore, they were resistant to penicillin, ampicillin, florfenicol, and tetracycline, and had elevated MICs for gentamicin, streptomycin, neomycin, and sulfisoxazole. Based on the AST results, these three isolates were selected for whole-genome sequencing to determine the genetic basis of their multidrug-resistance (MDR) phenotypes. The DNA was prepared by phenol-chloroform extraction. Closed genomes were obtained by hybrid assembly of Illumina MiSeq and Oxford Nanopore MinION reads. Sequence analysis revealed the presence of a Tn7406-like integrative and conjugative element (ICE) in all three isolates containing the antimicrobial resistance genes *erm(T)*, *mef(C)*, *mph(G)*, *floR*, *catA3*, *aac(3)-IIa*, *aph(3'')-Ib*, *aph(3')-Ia*-like, *tet(Y*)-like, and *sul2*. Isolate Mh191452 harbored an additional second copy of the *floR* gene within the Tn7406-like ICE. In addition, isolate Mh190176 carried a 9,226 bp plasmid with two copies of the *bla*ROB-1 gene, whereas the other two isolates each harbored a 4,614 bp plasmid (100% pairwise identity) with one *bla*ROB-1 gene. The detection of the macrolide resistance genes *erm(T)*, *mef(C)*, and *mph(G)* together with other resistance genes on a MDR-mediating ICE in bovine *M. haemolytica* shows that these isolates are already resistant to phenicols, penicillins, tetracyclines, and macrolides, which are regularly used for treating BRD. Due to the risk of limited therapeutic options, pathogen identification and subsequent AST is essential to ensure the efficacy of the antimicrobial agents applied to control BRD in cattle herds.

<footnote n="1">¹Institute of Microbiology and Epizootics, Centre for Infection Medicine, School of Veterinary Medicine, Freie Universität Berlin, Berlin, Germany</footnote>

<footnote n="2">²Veterinary Centre for Resistance Research (TZR), School of Veterinary Medicine, Freie Universität Berlin, Berlin, Germany</footnote>

<footnote n="3">³Federal Office of Consumer Protection and Food Safety (BVL), Berlin, Germany</footnote>
# Integration of transferable hybrid plasmids conferring resistance to carbapenem, colistin, and tigecycline in *Klebsiella pneumonia* isolated from house fly

Xiaoming Wang

MOE Joint International Research Laboratory of Animal Health and Food Safety, Centre for Risk Assessment of Veterinary Drug Residue and Antimicrobial Resistance, College of Veterinary Medicine, Nanjing Agricultural University, Nanjing, China

**Objectives:** To investigate the mechanisms for the formation of hybrid plasmids conferring resistance to carbapenem, colistin and tigecycline during the conjugation process, and to further characterize the transferability and stability of the newly formed multidrug resistant plasmids.

**Methods:** The locations of antimicrobial resistance genes on the plasmids were determined using S1-PFGE and Southern blot assay. The detailed genetic structures for these plasmids were depicted by a hybrid sequencing strategy using Illumina short-read and MinION long-read technology.

**Results:** We identified one *Klebsiella pneumoniae* isolate KP53 containing *bla*NDM-1, *mcr*-8, and *tmexCD1-toprJ1* on different plasmids. More specifically, these clinically important genes could be co-transferred through IS26 and *ltrA* mediated plasmid fusion to clinical isolates during conjugation under colistin selection, and then the recipient strains became carbapenemor tigecycline-resistant strains. The transferability and stability of these hybrid multi-drug resistance plasmids depend on the bacterial host and the presence of antibiotics.

**Conclusion:** This is the first time to report the forming of hybrid plasmid conferring resistance to carbapenems, colistin, and tigecycline. The biological advantages of the fusion plasmid indicated that the fusion event presumably plays a potential role in the dissemination of AMR. Considering the strain isolated from house fly, therefore, from a “One Health” perspective, further evolution and adaptation of these hybrid plasmids may facilitate their emergence and spread, which is of great concern for clinical therapy.

<figure><img src="image_54.png" /><figcaption>**Figure 1.** The integration of hybrid plasmids.</figcaption></figure>
# Emergence of high-level tigecycline resistance due to the amplification of *tet*(A) gene variants in clinical carbapenem-resistant *Klebsiella pneumoniae*

Xiang-Dang Du<fnref n="1" />*, Chunyan Xu<fnref n="1" />, Hong Yao<fnref n="1" />, Chenglong Li<fnref n="1" />, Runhao Yu<fnref n="1" />, Jinfeng Zhao<fnref n="1" /> and Stefan Schwarz<fnref n="3" />,⁴

The emergence of various genes mediating tigecycline resistance poses a significant risk to public health safety. Since the description of *tet*(X4), which was located on a plasmid and mediated high-level resistance to tigecycline, numerous studies have been conducted on tigecycline resistance. In previous reports, *tet*(A) variants have been considered to mediate low-level resistance to tigecycline, which received much less attention in clinical research. In this study, we found a greatly high rate (62.1%, 998/1,607) of *tet*(A) variants carriage in 1,607 carbapenem-resistant *Klebsiella pneumoniae* isolates from Henan Province, China. Moreover, we confirmed that high-level tigecycline resistance could be rapidly produced by the amplification of *tet*(A) variants in these isolates. The amplification and overexpression of *tet*(A) variants were verified by the determination of gene copy numbers and qRT-PCR. Through the analysis of the raw sequencing data and the plasmid mapping depth, we found that the *ΔtnpA* homologous sequence of Tn1721 supports the amplification of the region that harbors the *tet*(A) variants and forms a large number of repeat arrays through translocatable units (TUs). Moreover, the epidemiological analysis of *tet*(A) variant-carrying structures among 1,607 clinical carbapenem-resistant *Klebsiella pneumoniae* isolates indicated that the TU structure formed by the homologous sequence *ΔtnpA* is widely present. These results indicated that the presence of tigecycline resistance-mediating *tet*(A) variants in this clinical pathogen represents a greater health concern than initially thought and should be monitored consistently.

## References

[1] Yao H, Qin S, Chen S, Shen J, & Du XD (2018). *Lancet Infect Dis.* 18(1):25.

[2] Yu R, Chen Z, Li D, Schwarz S, Wang X, Du XD (2022). *Microbiol Spectr.* 10(3):e0049622.

[3] He T, Wang R, Liu D, Walsh TR, Zhang R, Lv Y, Ke Y, Ji Q, Wei R, Liu Z, Shen Y, Wang G, Sun L, Lei L, Lv Z, Li Y, Pang M, Wang L, Sun Q, Fu Y, Song H, Hao Y, Shen Z, Wang S, Chen G, Wu C, Shen J, Wang Y (2019). *Nat Microbiol.* 4(9):1450-1456.

<footnote n="1">¹International Joint Research Center of National Animal Immunology, College of Veterinary Medicine, Henan Agricultural University, Zhengzhou, 450046, P. R. China.</footnote>

<footnote n="2">²Institute of Microbiology and Epizootics, Centre for Infection Medicine, School of Veterinary Medicine, Freie Universität Berlin, Berlin, Germany.</footnote>

<footnote n="3">³Veterinary Centre of Resistance Research (TZR), Freie Universität Berlin, Berlin, Germany.</footnote>

<footnote>*Corresponding author. E-mail: xddu@henau.edu.cn</footnote>
# Are ionophore anticoccidials drivers for selection of resistance to clinical antibiotics on Norwegian poultry farms?

Rikki FREDERIKSEN<fnref n="1" />, Karin LAGESEN<fnref n="1" />, Roger SIMM<fnref n="2" />, Silje GRANSTAD<fnref n="1" /> and Anne Margrete URDAHL<fnref n="1" />

Coccidiosis, a disease caused by intestinal infection by protozoa belonging to the genus *Eimeria*, is a significant challenge to poultry health and welfare. The economic impact of coccidiosis in modern poultry production is substantial. Ionophores were introduced as anticoccidials in the 1970s, and in-feed prophylactic combinations of ionophores and synthetic chemicals are currently the most widely applied means of coccidiosis control in major poultry producing countries. Ionophores are ion carriers display both anticoccidial and antibacterial activity. Due to heavy use, widespread resistance to ionophores has been reported in both *Eimeria* and in indicator bacteria such as *Enterococcus faecium*. In 2012, it was observed that ionophore-resistance genes, later termed *narAB*, and resistance genes to last resort glycopeptide drugs (vancomycin) in *E. faecium* isolates from Swedish broilers could be co-transferred in the laboratory by bacterial conjugation [1]. Such a co-selection of ionophore and antibiotic-resistance offered an explanation for the concerning persistence of glycopeptide-resistant *E. faecium* in poultry and poultry farmers despite these glycopeptide drugs having been banned for more than two decades. The *narAB* resistance operon and its co-transfer has been found to be induced by exposure to ionophores [3]. Recently, *E. faecium* isolates from Dutch broilers have been found to carry plasmids encoding *narAB* along with resistance genes to antibiotics such as macrolides, tetracyclines and aminoglycosides [3]. Together these findings implies that the use of ionophores might drive the dissemination of resistance to medically important antibiotics in poultry. As a part of a bigger project, we have in this pilot study sequenced 20 *E. faecium* isolates from Norwegian broilers and turkeys to describe the phylogenetic distribution of AMR genes to explore whether bacteria from Norwegian poultry farms carry the potential for an ionophore-driven persistence of antibiotic resistance.

<figure><img src="image_55.png" /></figure>

Proposed model for co-selection of ionophore- and antibiotic-resistance in poultry-associated bacteria.

## References

[1] O. Nilsson, C. Greko, B. Bengtsson, and S. Englund (2012). *J Appl Microbiol.* Apr;112(4):716-22

[2] A-O. Naemi, H. Dey, N Kiran, S. T. Sandvik, J. S. Slettemeås, L. L. Nesse *et al.* (2019). *Front. Microbiol* 11:104

[3] M. G. Pikkemaat, M. L. B. A. Rapallini, J. H. M. Stassen, M. Alewijn *et al.* (2019). *WFSR Report 2022.005*

<footnote n="1">¹Norwegian Veterinary Institute, Ås, Norway</footnote>

<footnote n="2">²Institute of Oral Biology, University of Oslo, Oslo, Norway</footnote>
# Identification of the novel Macrolide-Lincosamide-Streptogramin B resistance gene erm(54) in a porcine LA-MRSA ST398

Henrike KRÜGER-HAKER<sup>1,2</sup>, Xing JI<sup>3</sup>, Dennis HANKE<sup>1,2</sup>, Anne-Kathrin SCHINK<sup>1,2</sup>, Stefan FIEDLER<sup>4</sup>, Heike KASPAR<sup>4</sup>, Yang WANG<sup>5</sup>, Stefan SCHWARZ<sup>1,2,5</sup>, Congming WU<sup>5</sup> and Andrea T. FEßLER<sup>1,2</sup>

**Background and objectives:** Livestock-associated methicillin-resistant *Staphylococcus aureus* (LA-MRSA) can represent a health risk for humans, especially for those having frequent contact to livestock [1]. LA-MRSA are mainly associated with sequence type (ST) 398 in many parts of the world and often multiresistant to antimicrobial agents [1]. Here, 178 porcine LA-MRSA isolated in Germany from 2007 - 2019 were investigated for new antimicrobial resistance genes.

**Methodology:** Whole-genome sequencing on Illumina MiSeq and PacBio Sequel II platforms was followed by hybrid assembly and sequence analysis. Plasmid pHKS3860 was transferred into *S. aureus* RN4220 via electrotransformation. Antimicrobial susceptibility testing via broth microdilution and agar disc diffusion was performed according to CLSI standards [2] to confirm the functionality of *erm*(54). An *erm*(54)-specific PCR assay was developed and applied to 30 macrolide-resistant staphylococcal isolates, which harbored next-related *erm* genes.

**Results:** A novel Macrolide-Lincosamide-Streptogramin B (MLS<sub>B</sub>) resistance gene, *erm*(54), was detected on the non-conjugative plasmid pHKS3860 of 36,929 bp in a porcine LA-MRSA ST398. The gene encoded a 23S rRNA methylase of 245 amino acids (aa) that was next-related to Erm(B) (72%). Moreover, *erm*(54) was expressed constitutively. A complex regulatory region composed of a small reading frame for a 30 aa protein and seven pairs of inverted repeats, which can form varying mRNA secondary structures, was detected upstream of *erm*(54). The transferred *erm*(54) caused a distinct increase in the minimal inhibitory concentrations of MLS<sub>B</sub> antibiotics in *S. aureus* RN4220. The new PCR assay detected *erm*(54) in the original strain and the transformants carrying pHKS3860, but none of the next-related *erm* genes available to us. Copper, mercury and cadmium resistance genes as well as an *ica* cluster for biofilm formation were also found on plasmid pHKS3860.

**Conclusion:** The new transferable and functionally active MLS<sub>B</sub> resistance gene *erm*(54) was identified in a porcine LA-MRSA ST398 isolate from Germany. The co-location of *erm*(54) on a plasmid with heavy metal resistance genes may increase the risk for co-selection under selection pressure imposed by heavy metals in animal feed or the environment. Due to this possibility of co-selection and the zoonotic potential, *erm*(54)-carrying isolates might pose a public health threat in the future.

## References

[1] P. Butaye, M. A. Argudín and T. C. Smith (2016). *Curr Clin Microbiol Rep* 3:19-31.

[2] Clinical and Laboratory Standards Institute (CLSI) (2022): CLSI supplement M100, 32nd Ed.

<footnote n="1"><sup>1</sup>Institute of Microbiology and Epizootics, Centre for Infection Medicine, School of Veterinary Medicine, Freie Universität Berlin, Berlin, Germany</footnote>

<footnote n="2"><sup>2</sup>Veterinary Centre for Resistance Research (TZR), School of Veterinary Medicine, Freie Universität Berlin, Berlin, Germany</footnote>

<footnote n="3"><sup>3</sup>Jiangsu Key Laboratory for Food Quality and Safety, State Key Laboratory, Cultivation Base of Ministry of Science and Technology, Institute of Food Safety and Nutrition, Jiangsu Academy of Agricultural Sciences, Nanjing, China</footnote>

<footnote n="4"><sup>4</sup>Federal Office of Consumer Protection and Food Safety (BVL), Berlin, Germany</footnote>

<footnote n="5"><sup>5</sup>Key Laboratory of Animal Antimicrobial Resistance Surveillance, MARA, College of Veterinary Medicine, China Agricultural University, Beijing, China</footnote>
# Allicin served as a New Delhi metallo-β-lactamase inhibitor : a new strategy to overcome superbugs

Xi LV, Zhibin YE₁, Liping WANG

MOE Joint International Research Laboratory of Animal Health and Food Safety, Centre for Risk Assessment of Veterinary Drug Residue and Antimicrobial Resistance, College of Veterinary Medicine, Nanjing Agricultural University, Nanjing, China

The emergence and spread of carbapenem-resistant Enterobacteriaceae (CRE), mediated by metallo-β-lactamases (MBLs) like NDM, is a global public health problem. It is becoming a new consensus repurposing approved drugs or natural compounds as potential MBLs inhibitor to restore the efficacy of carbapenem. Previous studies have demonstrated that allicin, the main effective ingredient in garlic, has many pharmacological effects including antibacterial, anti-inflammatory, antiparasitic, anti-tumor, and treatment of cardiovascular and cerebrovascular diseases and been used in the treatment and prevention of a wide variety of ailments. In this study, we focus on investigating the effect of allicin on CRE as well as its inhibitory activity on NDM enzyme. Shown by the following figure, allicin displayed significant antibacterial activity against NDM-carrying *E. coli* strains with MIC values from 16-64 µg mL⁻¹ and presented excellent synergistic activity with meropenem (FICI=0.357). In addition, we identified that allicin is an effective inhibitor of the NDM enzyme with 50% inhibiting concentration (IC50) was 18.63 ± 1.39 µM. The equilibrium dissociation constant (Kd) to NDM was 0.14 ± 0.087 µM, which was determined by microscale thermophoresis. AutoDocking analysis indicated that allicin inactivates NDM by binding to the active site Cys208. And the zinc sensitivity experiments showed that the binding of allicin to NDM resulted in the release of Zn(II). Finally, a mouse leg muscle model infected with NDM-carrying *E. coli* proved that the combination of allicin and meropenem significantly reduced the bacterial load. In conclusion, our findings demonstrate that the combination of allicin and meropenem, a carbapenem antibiotic, has therapeutic potential to address the clinical challenge of MBL-positive CRE strains.

<figure><img src="image_56.png" /><figcaption>**Fig. 1.** Allicin served as a NDM inhibitor.</figcaption></figure>

## References

[1] Yong D, Toleman M A, Giske C G, Cho H S, Sundman K, et al. (2009). *Antimicrob Agents Chemother* 53(12):5046-5054.

[2] Bush, K., Bradford, P.A (2019). *Nat Rev Microbiol* 17:295-306.
<figure><img src="image_57.png" /></figure>

# The AcrAB Efflux Pump Confers Intrinsic Multidrug Resistance and Self-Protection against Stilbenes in *Photorhabdus laumondii*

Linda Hadchity<fnref n="1" />,<fnref n="2" />, Anne Lanois<fnref n="1" />, Jessica Houard<fnref n="3" />, Amaury Payelleville⁴, Maxime Gualtieri<fnref n="3" />, Alain Givaudan<fnref n="1" /> and Ziad Abi Khattar<fnref n="2" />

The global shortage of new antimicrobials brought us to antimicrobial resistance which has emerged to last-resort antibiotics. The Resistance-nodulation-division (RND)-type AcrAB-TolC efflux pump contributes to multidrug resistance in Gram-negative bacteria. Recently, the bacterium *Photorhabdus laumondii* TT01 has emerged as a goldmine for novel anti-infective drug discovery. Outside plants, *Photorhabdus* is the only Gram-negative known to produce stilbene-derivatives including 3,5-dihydroxy-4-ethyl-trans-stilbene and 3,5-dihydroxy-4-isopropyl-trans-stilbene (IPS). IPS is a bioactive polyketide which received considerable attention, mainly because of its antimicrobial properties, and is currently in phase III clinical trials as a topical treatment for psoriasis. To date, little is known about how *Photorhabdus* survives in the presence of stilbenes. We combined genetic and biochemical approaches to assess whether AcrAB efflux pump exports stilbenes in *P. laumondii*. We demonstrated that the wild type (WT) exerts an antagonistic activity against its derivative Δ*acrA* mutant, and that is able to outcompete it in a dual-strain co-culture assay [1, 2]. The Δ*AcrA* mutant also showed high sensitivity to 3,5-dihydroxy-4-ethyl-trans-stilbene and IPS as well as decreased IPS concentrations in its supernatant comparing to the WT [2]. We report here a mechanism of self-resistance against stilbene derivatives of *P. laumondii* TT01, which enables these bacteria to survive under high concentrations of stilbenes by extruding them out via the AcrAB efflux pump [2]. Our findings emphasize the poly-specificity and versatility of AcrAB to export diverse native antimicrobial compounds in Gram-negative bacterial species and may represent a promising target feature to improve the control of resistance to innovative antibiotics.

## References

[1] L. Hadchity, et al. (2021). Environmental microbiology reports 13:637-48.

[2] L. Hadchity, et al. (2023). Research in Microbiology.

<footnote n="1">¹Laboratoire Diversité, Génomes et Interactions Microorganismes-Insectes (DGIMI), INRAE / Université de Montpellier, Montpellier, France</footnote>

<footnote n="2">²Laboratory of Georessources, Geosciences and Environment (L2GE), Faculty of Sciences 2 / Lebanese University, Fanar, Lebanon</footnote>

<footnote n="3">³Nosopharm, Nîmes, France 4 Cellular and Molecular Microbiology, Faculté des Sciences / Université Libre de Bruxelles, Gosselies, Belgium</footnote>
<figure><img src="image_58.png" /></figure>

Session 4 - Understanding the connection of antimicrobial resistance between
Animals and Humans

P65

The role of sub-inhibitory antibiotic pollution on MGEs in the environment

Concepcion Sanchez-Cid<fnref n="1" />, Timothy M. Vogel<fnref n="1" />

Antibiotic residues resulting from anthropogenic activities are released to the environment and often at concentrations that are under the bacterial growth inhibition threshold. Sub-inhibitory concentrations of antibiotics, despite being too low to inhibit overall bacterial growth in environmental communities, might impose a selective pressure on some community members and induce a response in environmental bacteria. Consequently, the presence of antibiotic residues at sub-inhibitory concentrations in the environment could lead to the mobilization of antibiotic resistance genes (ARGs) in mobile genetic elements (MGEs) and the dissemination of these MGEs in environmental settings and to the human microbiome through horizontal gene transfer (HGT). Although increasing evidence points to a potential risk for human health associated to environmental sub-inhibitory antibiotic pollution, the magnitude of this risk still needs to be elucidated. Our research addressed the role of sub-inhibitory concentrations of antibiotics on the mobilization of environmental ARGs into MGEs such as class 1 integrons and plasmids, and the resulting dissemination potential of these MGEs in the environment and to the human microbiome. We used a combination of culture-based and metagenomics approaches to evaluate the selective potential of sub-inhibitory concentrations of a range of antibiotics (e.g., gentamicin, ciprofloxacin) with or without heavy metals and microplastics on MGEs and their associated ARGs. We evaluated the influence on the metagenomes of the river microbial community in order to delineate possible mechanisms underlying the response of these MGEs to sub-inhibitory pollution. Antibiotics at sub-inhibitory concentrations increased the mobilization of antibiotic resistance genes in class 1 integrons and the dissemination potential of these genes. Our research shows molecular evidence of the impact of antibiotics at sub-inhibitory concentrations on environmental MGEs and the mechanisms underlying the molecular response to sub-inhibitory antibiotic pollution.

<footnote n="1">¹Université de Lyon, Université Claude Bernard Lyon 1, UMR CNRS 5557, UMR INRAe 1418, VetAgro Sup, Ecologie Microbienne, équipe BEER, F-69622 Villeurbanne, France.</footnote>
# Investigative genomics on highly antibiotic resistant *Pseudomonas* isolated from rainbow trout (*Oncorhynchus mykiss*)

Kenny OBERLÉ¹, Gaëlle BEDNAREK¹, Claude RISPE¹ and Ségolène CALVEZ¹

¹BIOEPAR UMR 1300, Oniris - INRAE, Nantes, France

Open aquaculture systems are farming areas with strong relationships between food-producing fishes and their aquatic environment. Aquatic animals are exposed to several physico-chemical variations and to human or terrestrial animal discharges with chemical and microbiological contaminants. Fishes host autochtonous bacteria which can encounter allochtonous ones brought by the river inflow in farms. *Pseudomonas* is a bacterial genus that is ubiquitous in many environments and often isolated from fish such as rainbow trout and their environment¹,². We previously identified *Pseudomonas* isolates collected on rainbow trout fillets from two different farms, eight of which showing high minimum inhibitory concentrations (MICs) to one or several antibiotics³. Seven out of eight isolates showed multiple high MICs to oxytetracycline (64 to 256 µg.mL⁻¹), florfenicol (512 to >1,024 µg.mL⁻¹), colistin (512 to >1,024 µg.mL⁻¹) and trimethoprim/sulfamethoxazole (128/2432 to 512/9728 µg.mL⁻¹).

In this study, we focused on genome architecture of these eight isolates with high MICs to establish relationship between phenotype and genotype, and studied the presence of mobile genetic elements (MGEs) to better understand if these genes can be transferred horizontally. After DNA isolation (DNeasy Blood & Tissue Kit by Qiagen and Wizard® Genomic DNA Purification Kit by Promega) of each *Pseudomonas* isolate, genomic DNA was sequenced using Illumina (short reads) and Oxford Nanopore (long reads) technologies. Hybrid genomes assembly were performed using UniCycler then scaffolding of all assemblies at contig levels were carried out and compared to a closely related genome used as a reference sequence (*P. fluorescens* SBW25). Subsequent searches of antibiotic resistance genes have been done using the Resistance Gene Identifier tool of the Comprehensive Antibiotic Resistance Database (CARD) and completed with the ResFinder tool. MGEs as insertion sequences, transposons, plasmids have been investigated using ISfinder and the plasmid database PLSDB when applicable. Prophage sequences were also searched using PHASTER.

The sequenced genomes showed a total length ranging between 6Mbp and 6.5Mbp. In-depth analyses of these genomes established that most of high MICs could be related to at least one known antibiotic resistance gene with *tetY* for oxytetracycline, *floR* for florfenicol and *sul2* for trimethoprim/sulfamethoxazole. No *mcr* gene was detected for the two isolates with high MICs to colistin. Additional genes related to unassessed phenotype resistance have been identified such as *AbaQ* assuming to encode for an efflux pump mediating quinolone resistance or *aph(6)-Id* and *aph(3')-Ib* already known for coding aminoglycoside phosphotransferases. A wide range of insertion sequences and transposons were also detected in all isolates such as IS66, IS5, Tn3 or Tn5 families. Every *Pseudomonas* isolate comprised prophage sequences in several regions of their genomes with intact or incomplete match with common phages. Our results highlight potential genetic bases for atypical phenotypes found in *Pseudomonas* hosted by rainbow trout. This study reinforces the interest to better study bacterial whole genomes to provide new elements for understanding the role of horizontal gene transfer in their architecture but also the spreading of antibiotic resistance.

## References

1. Dominguez, M. et al. (2019). *Front. Microbiol.* **10**, 748.

2. Akinbowale, O. L. et al. (2007). *Int. J. Antimicrob Agents* **30**, 177–182 (2007).

3. Oberlé, K. et al. (2022). *J Appl Microbiol* **132**, 1738–1750.
# Administration of oxytetracycline and resistance selection in the different digestive segments: determination of selective minimum concentrations and impact on the microbiota and resistome in pigs

Pedro H. IMAZAKI¹, Bertille VOISIN¹, Clémentine DE SMET¹, Marlène Z. LACROIX¹, Felipe RAMÓN PORTUGAL¹, Nathalie ARPAILLANGE¹, Alain BOUSQUET-MÉLOU¹, Aude FERRAN¹, Véronique DUPOUY¹ and Delphine BIBBAL¹

¹INTHERES, University of Toulouse, INRAE, ENVT, Toulouse, France

After oral administration, a fraction of antibiotics binds to the digestive matrix. Nevertheless, the free or active concentrations exert a pressure of selection on the microbiota, favouring resistance selection. Therefore, the first objective of this study was to determine the minimum selective concentration (MSC) of oxytetracycline (OTC); the MSC being the lowest concentration of an antibiotic conferring a competitive advantage to a resistant strain upon an isogenic susceptible strain. The second objective was to characterise the impact of OTC administration on the microbiota and resistome in the different digestive segments of pigs.

Co-cultures of an OTC-susceptible *Escherichia coli* strain and a resistant isogenic strain were performed in the presence of different concentrations of OTC in Muller-Hinton broth (MHB) and sterilised intestinal contents (SIC) of the jejunum, cecum and rectum of pigs. These strains were counted at different times, permitting to calculate the selection coefficient at each concentration of OTC and, by extrapolation, the MSC [1]. Subsequently, a therapeutic dose of OTC (20 mg/kg bw) was administered orally to eight piglets. The contents of the digestive segments were harvested 6 (n = 4) and 24 hours (n = 4) after treatment. The control group consisted of four untreated animals. Tetracycline resistance genes were quantified by qPCR. Microbial diversity was assessed by sequencing the V3-V4 region of the gene encoding 16S rRNA. Finally, OTC was quantified by UPLC-UV to determine the total concentrations in the different digestive segments.

MSC was 0.025 µg/mL in BMH and increased to 0.26, 0.72, and 2.4 µg/mL in the jejunum, cecum, and rectum, respectively. The binding between antibiotics and constituents of the digestive matrix may have influenced the free (active) antibiotic fraction, this bond being stronger in the distal digestive tract [2]. In addition, the interaction between bacteria and constituents of the digestive matrix may have influenced their sensibility to antibiotics [3]. Along the digestive tract, the taxonomic composition of the microbiota changed, and the abundance of resistance genes increased, but the antibiotic treatment did not impact the microbiota. Finally, 6 h after administration, OTC concentration was 49.7, 65.9 and 5.7 µg/g, and 12 h after administration the concentrations were >LOQ, 18.6 and 97.9 µg/g in the contents of jejunum, cecum and rectum, respectively. We will determine the active OTC concentrations using bactericidal curves produced in MHB and SIC.

In conclusion, this study provides new data for the risk assessment of antibiotic resistance in the digestive environment. In addition, we plan to improve our *in vitro* model to consider the effect of gut microbiota on MSC. Finally, the developed methodology could also be applied to the risk assessment of other antibiotics or other matrices (e.g. environmental samples).

## References

[1] E. Gullberg, S. Cao, O.G. Berg, C. Ilbäck, L. Sandegren, et al. (2011). *PLOS Pathog* 7:e1002158.

[2] A. Ferran, D. Bibbal, T. Pellet, M. Laurentie, M. Gicquel-Bruneau, et al. (2013). *Int J Antimicrob Agents* 42:429-435.

[3] Q. Vallé, B. B. Roques, A. Bousquet-Mélou, D. Dahlhaus, F. Ramón Portugal, et al. (2021). *Front Microbiol* 12: 671376.
# Development of a *Salmonella enterica* Plasmid Transfer Gene Database and Associated Analysis Tools

Jing HAN<fnref n="1" />, Hailin TANG³, Suad ALGARNI<fnref n="1" />,<fnref n="2" />, Shaohua ZHAO<fnref n="4" />, and Steven L. FOLEY<fnref n="1" />,<fnref n="2" />

Antimicrobial resistant (AMR) enteric pathogens, including *Salmonella*, has become a significant concern due to potential treatment failures. Often, AMR genes are encoded on transmissible plasmids and the conjugal transfer of resistance plasmids can lead to the dissemination of AMR among bacterial populations. Type IV secretion systems (T4SSs), which are integral parts of the conjugation process in enteric bacteria, are encoded on the transfer (tra) regions of plasmids. Better understanding of the diversity and functionality of the T4SS genes that can facilitate the development strategies to control and prevent spreading of temergence of multidrug-resistant pathogens. To facilitate the analyses of these transfer-associated genes, the current project was undertaken to identify the transfer associated genes that are associated with conjugal secretion systems in key groups of AMR plasmids. The information of these transfer-associated genes was extracted from GenBank and incorporated into a Plasmid Transfer Gene Database. After the database set up, two comparison tools were developed. One of the tools named “the Plasmid Transfer Factor Assessment” is used to predict the transfer genes present in the specific plasmid location and their sequence similarity to the reference genes. The other tool was named the “Plasmid Transfer Factor Comparison tool”, which can be used to facilitate the comparison transfer genes of plasmids from multiple sequence files. To assess the databases, plasmid and whole genome sequencing (WGS) data identified from GenBank and previous WGS experiments in our lab were extracted and used to assess the tools. Overall, when plasmids were identified with PlasmidFinder, their T4SS-associated genes were typically identified with the database tools. In some cases, there were significant overlaps such as with genes from IncBO and IncK plasmids, thus the database was updated to combine the genes from these plasmid types into a renamed IncB/O/K group. The current updated database contains 337 genes representing the T4SSs from 13 different plasmid groups. The plasmid transfer database and tools proved very useful technique for evaluating the different plasmid types and their T4SSs. The ability to predict all T4SS genes that are present can improve our understanding how conjugative plasmids contribute to the dissemination of antimicrobial resistance genes.

<footnote n="1">¹Division of Microbiology and ³Division of Bioinformatics and Biostatistics, FDA National Center for Toxicological Research, Jefferson, Arkansas, USA</footnote>

<footnote n="2">²Cell and Molecular Biology Program, University of Arkansas, Fayetteville, Arkansas, USA</footnote>

<footnote n="4">⁴Office of Research, FDA Center for Veterinary Medicine, Laurel, Maryland, USA</footnote>
# Screening for CPE in livestock in the Netherlands using multiplex real-time PCR

Yvon GEURTS<fnref n="1" />, Bart WULLINGS<fnref n="2" /> and Kees VELDMAN<fnref n="1" />

**Background:** Carbapenemase-producing *Enterobacteriacea* (CPE) are an increasing problem worldwide. As a consequence monitoring of CPE in livestock and meat became mandatory in 2021 after the new European legislation on antimicrobial resistance monitoring in livestock and meat came into force. The prescribed method for detection of CPE is selective culturing, but this method has its limitations [1]. For this reason a reliable and cost-effective method for rapid and sensitive screening of CPE in fecal samples is desired. In this study a set of two multiplex real-time PCR assays was developed.

**Method:** Previously published primers were combined with in-house designed primers to set up the multiplex real-time PCR assays using SybrGreen chemistry. The first multiplex real-time PCR covers the most predominant carbapenemase genes *bla*<sub>NDM</sub>, *bla*<sub>KPC</sub>, *bla*<sub>VIM</sub>, *bla*<sub>IMP</sub> and *bla*<sub>OXA</sub>. The second multiplex real-time PCR covers the more rarely detected genes *bla*<sub>IMI</sub>, *bla*<sub>FRI</sub> and *bla*<sub>FLC</sub> earlier reported in *Enterobacter cloacae* complex isolates from imported fish and shrimps [2]. This set of PCR's is completed with an amplification control PCR to test for inhibitory factors in the DNA. The scope of the first PCR was focused on the gene variants reported in European livestock [3] (table 1). Primers are selected for each gene group to amplify all gene variants listed in table 1. Subsequent sequencing of the amplicons is necessary to determine the specific gene variants.

<table><caption>**Table 1** Carbapenemase gene variants found in European livestock</caption><thead><tr><th>Target</th><th>NDM</th><th>KPC</th><th>VIM</th><th>IMP</th><th>OXA</th></tr></thead><tbody><tr><td>Gene variants</td><td>NDM-1<br>NDM-4<br>NDM-5<br>NDM-7<br>NDM-9<br>NDM-17</td><td>Not specified</td><td>VIM-1</td><td>IMP-1<br>IMP-2<br>IMP-27</td><td>OXA-48/OXA-48-like<br>OXA-58<br>OXA-181</td></tr></tbody></table>

For CPE monitoring of livestock in the Netherlands pools of five selective enrichment samples are tested with the described set of PCR's. When a pool is tested positive with a PCR, the individual samples of the pool will be tested. Additionally, carbapenem resistant isolates grown from positive enrichment samples are tested with the PCR-set. The presence of carbapenem genes in CPE-suspected isolates is confirmed by sequencing. To date, no CPE were detected in faecal samples of livestock in the Netherlands.

## References

[1] A European multicenter evaluation study to investigate the performance on commercially available selective agar plates for the detection of carbapenemase producing Enterobacteriaceae; C. Dierikx et al; Journal of Microbiological Methods, Volume 193, 2022, 106418, ISSN 0167-7012, https://doi.org/10.1016/j.mimet.2022.106418

[2] Novel Carbapenemases FLC-1 and IMI-2 Encoded by an Enterobacter cloacae Complex Isolated from Food Products. MSM Brouwer et al; Antimicrob Agents Chemother. 2019 May 24;63(6)

[3] Carbapenem-resistant Enterobacteriaceae in wildlife, food-producing, and companion animals: a systematic review; R. Köck et al; Clin Microbiol Infect 2018;24:1241

<footnote n="1">¹Wageningen Bioveterinary Research (WBVR), Lelystad, the Netherlands</footnote>

<footnote n="2">²Wageningen Food Safety Research (WFSR), Wageningen, the Netherlands</footnote>
<figure><img src="image_59.png" /></figure>

# Onsite and rapid detection of tigecycline-resistant hypervirulent *Klebsiella pneumoniae* using CRISPR/Cas12a based assay

Chenglong LI, Hong YAO, Chunyan XU, Yishuai WU, Jinfeng ZHAO and Xiang-Dang DU

International Joint Research Center of National Animal Immunology, College of Veterinary Medicine, Henan Agricultural University, Zhengzhou, China

Tigecycline-resistant hypervirulent *Klebsiella pneumoniae* (HvKP) is a notorious pathogen that poses a significant threat to public health, as it could cause severe infections with high morbidity and mortality among young and healthy individuals. Currently, a positive string test has been primarily used to identify HvKP isolates. However, this method with an accuracy of 90% is more suitable for hypermucoviscous phenotype. An accurate and rapid assay to identify tigecycline-resistant HvKP infections is needed for optimal clinical care and infection control efforts. Therefore, in the present study, CRISPR/Cas12a based method was developed for onsite detection of tigecycline-resistant HvKP based on the virulence biomarkers *iucA*, *iroB*, *peg-344*, *rmpA* and *rmpA2*, and the resistance gene *tet*(A). Upon optimization, the CRISPR/Cas12a based method detected 1 cfu/reaction of tigecycline-resistant HvKP with high accuracy and specificity, and the whole procedure took 60 min including DNA rapid extraction. Moreover, the whole process can be performed with a portable block heater, and the detection results can be observed with the naked eye. The developed CRISPR/Cas12a assay could be used as a diagnostic tool with onsite, portable, rapid, and simple for the practical identification of tigecycline-resistant HvKP infections.

<figure><img src="image_60.png" /><figcaption>**Fig. 1.** Simultaneous detection of the genes *iucA*, *iroB*, *peg-344*, *rmpA* and *tet*(A) using the developed CRISPR/Cas12a method. Strains with 10⁶, 10⁵, 10⁴, 10³ and 10² cfu/mL were used in this study. A positive symbol (+) indicates that the extracted DNA was added as the template, while a negative symbol (-) implies that nuclease-free water was used as the negative control.</figcaption></figure>

## References

[1] J.S. Chen, E. Ma, L.B. Harrington, M. Da Costa, X. Tian, et al. (2018). *Science* 360: 436-439.

[2] H. Yao, S. Qin, S. Chen, J. Shen and X.-D. Du (2018). *Lancet Infect. Dis.* 18: 25.
# Conifer needles as a passive air sampler for antibiotic resistance genes (ARGs) detection emitted during pig manure spreading

Samantha LECLERC<fnref n="1" />, Paul GEORGE<fnref n="1" />,<fnref n="2" />, Marc VEILLETTE<fnref n="1" />, Nathalie TURGEON<fnref n="1" /> and Caroline DUCHAINE<fnref n="1" />,<fnref n="2" />,<fnref n="3" />

Bacterial antibiotic resistance was identified worldwide as an urgent global public health threat. Antibiotic resistance genes (ARGs) can be acquired and/or transferred by bacteria through different mechanisms. Due to the heavy usage of antibiotics in agricultural activity, ARGs are abundant in manure and can potentially be dispersed in the environment over significant distances by spreading activities on fields. ARGs propagation should be studied to better assess the antibioresistance risk. Biomonitoring of air is generally performed using active samplers. However, these devices are expensive, bulky and recover air for a relatively short time window. Passive samplers, such as conifer needles, could be used as an alternative for air monitoring. This project aims to assess the ARGs accumulation on conifer needles compared to active high flow sampling during pig manure field spreading. Four spreading activities of pig slurry were conducted on an experimental farm. Air samples were taken with two high-flow samplers, during swine manure spreading. Downwind (3 distances) and upwind samples were collected. Conifer needles, from trees located downwind of the sampling site were sampled before and after (24h) spreading activities. Conifer needles processing has been achieved according to George et al. [1]. Total bacteria concentration was quantified by quantitative PCR (qPCR) for the pig manure, the conifer needles, and the air samples. Chloroplasts were quantified in conifer needles (rcbL). One fecal indicator and one pig DNA indicator (phage vB_AviM_AVP and Sus domesticus cytochrome b gene) were assessed in conifer needles and air samples bioaerosol source confirmation. Manure samples were tested for the presence of 40 different ARGs [2]. Amongst the genes tested, 28 were detected and are associated with resistance to sulfonamides (2), tetracyclines (10), erythromycin (5), β-lactams (2), quinolones (2), aminoglycosides (3), and colistin (1). Mobile genetic elements (3) were also detected. These positive genes were used to analyse the conifer needles and the air samples with a high-throughput qPCR Smartchip platform (Takara). For both conifer needles and air samples, the most frequently detected ARGs were in accordance with what is observed in manure samples: tetracyclin (tet) and erythromycin (erm). The genes with the highest frequency in conifer needles were tetW and erm35 (72% and 14% respectively). In air samples tetW and ermB (44% and 33% respectively). Manure spreading from animal farms lead to dissemination of ARGs in the environment and can potentially represent a threat for human and animal health. Conifer needles could be a simple and affordable long-term sentinels to monitor ARGs dispersion through bioaerosols.

## References

[1] George PBL, Leclerc S, Turgeon N, Veillette M, Duchaine C. Conifer Needle Phyllosphere as a Potential Passive Monitor of Bioaerosolised Antibiotic Resistance Genes. Antibiotics (Basel). 2022;11(7); doi: 10.3390/antibiotics11070907.

[2] George PBL, Rossi F, St-Germain MW, Amato P, Badard T, Bergeron MG, et al. Antimicrobial Resistance in the Environment: Towards Elucidating the Roles of Bioaerosols in Transmission and Detection of Antibacterial Resistance Genes. Antibiotics (Basel). 2022;11(7); doi: 10.3390/antibiotics11070974.

<footnote n="1">¹Centre de l'Institut Universitaire de Cardiologie et de Pneumologie de Québec-Université Laval</footnote>

<footnote n="2">²Département de biochimie, de microbiologie et de bio-informatique de l'Université Laval, Québec, Canada</footnote>

<footnote n="3">³Canada Research Chair on Bioaerosols</footnote>
# MyMIC: a network for standardization of diagnostics, antimicrobial susceptibility testing and clinical interpretation in animal mycoplasmas

Anne V. GAUTIER-BOUCHARDON<fnref n="1" />, Maryne JAҮ<fnref n="2" />, Sara M. KLOSE<fnref n="3" />,
Annet HEUVELINK<fnref n="4" />, Inna LYSNYANSKY<fnref n="5" />, Miklós GYURANECZ<fnref n="6" />, Ana S. RAMIREZ<fnref n="7" />,
Marco BOTTINELLI<fnref n="8" />, Anne RIDLEY<fnref n="9" />, Anneke FEBERWEE<fnref n="4" />, Claire BECKER<fnref n="2" />,
and Florence TARDY<fnref n="1" />,<fnref n="2" />.

Mycoplasma is a bacterial genus that is often disregarded although it includes species that are of importance for both human and animal medicine. In animals, some species are major pathogens causing various diseases and significant economic losses in avian, porcine and ruminants sectors. In the absence of efficient vaccines, their control relies mainly on chemotherapy and thus mycoplasmas contribute largely to antibiotic use and hence to the general rise of resistance. Because of their intrinsic resistance to the broadly used β-lactams family and the increasing number of acquired resistances to other antimicrobial families [1,2], mycoplasmas must be subjected to an improved antimicrobial resistance surveillance. However, standard procedures, used for classic bacteria to assess the efficacy of antibiotics by in vitro antimicrobial susceptibility testing (AST), such as disk diffusion method, cannot be used for mycoplasmas, because of their fastidious growth and requirement for complex medium. Moreover, the lack of guidelines, quality control (QC) strains, clinical breakpoints or epidemiological cut-off (ECOFF) values for livestock mycoplasmas makes comparisons between studies and interpretation of results difficult or impossible. MyMIC is one of the six new collaborative networks funded by the Joint Programming Initiative on Antimicrobial Resistance (Diagnostics and Surveillance Networks – JPIAMR). It includes 22 laboratories from 18 countries working on animal Mycoplasma diagnostics and AST. The objective of this network is to make an inventory of the different methods used by laboratories for identification, culturing, determination of minimum inhibitory concentrations (MIC) per animal and Mycoplasma species, and detection of resistance mechanisms by molecular or genomic tools. The MIC values of the main families of antimicrobials used against some key pathogenic Mycoplasma species, obtained by identical or comparable methods, will also be collected and aggregated to determine if this data can be used to define first tentative ECOFF values. This network will lead to the drafting of guidelines for a standardized culturing, identification and determination of MIC for livestock Mycoplasma species. Moreover, it could be the start of new wet-lab research proposals for additional work to undertake in order to define QC strains, ECOFFs and clinical breakpoints.

**References:**

[1] S. Pereyre and F. Tardy (2021). *Antibiotics* 10: 1216. doi: 10.3390/antibiotics10101216.

[2] A.V. Gautier-Bouchardon (2018). *Microbiol Spectr* 6 (4). doi: 10.1128/microbiolspec.ARBA-0030-2018.

<footnote n="1">¹Anses, Ploufragan-Plouzané-Niort Laboratory, Ploufragan, France</footnote>

<footnote n="2">²UMR Animal Mycoplasmoses, Anses, VetAgro Sup, Lyon, France</footnote>

<footnote n="3">³University of Melbourne, Victoria, Australia</footnote>

<footnote n="4">⁴Royal GD, Deventer, The Netherlands</footnote>

<footnote n="5">⁵Kimron Veterinary Institute, Beit Dagan, Israel</footnote>

<footnote n="6">⁶Veterinary Medical Research Institute, Budapest, Hungary</footnote>

<footnote n="7">⁷Universidad de Las Palmas de Gran Canaria, Las Palma, Spain</footnote>

<footnote n="8">⁸Istituto Zooprofilattico Sperimentale delle Venezie, Buttapietra, Italy</footnote>

<footnote n="9">⁹Animal and Plant Health Agency, Woodham, United Kingdom</footnote>
# Application of MALDI-TOF MS for detection of carbapenem resistance in bacteria

**Katarzyna BIELIŃSKA<fnref n="1" />, Marta PIETRUK<fnref n="1" />, Renata KWIT<fnref n="2" /> and Dariusz WASYL<fnref n="1" />,<fnref n="2" />**

**Background and objectives:** Over the last few years, MALDI-TOF MS has been explored as a rapid antimicrobial resistance detection tool. This application is based on the fact that MALDI-TOF MS can easily monitor changes in antibiotic structure that can be related to bacterial resistance. In this study, we investigate specific mass shifts caused by hydrolysis in the β-lactam ring in three selected carbapenems, i.e., meropenem, ertapenem, and imipenem.

**Materials and methods:** Based on the MIC values 13 *Escherichia coli* (8 sensitive, 5 resistant to carbapenems) and 7 *Salmonella* spp. (4 sensitive, 3 resistant) strains were selected for the study. Fresh overnight nutrient agar cultures (Biomaxima) were employed for the MALDI-TOF MS analysis. Samples were prepared in duplicate, positive, negative, and antibiotic control was added to the study. An inoculation loop (1 µl) of bacterial culture was suspended in 20 µl of antimicrobial solutions (concentration 0,5 mg/ml) and incubated (37°C, 500 rpm, 30 min). Subsequently, the cultures were centrifuged (2 min, 13,000 g, room temperature), and 1 µl of the supernatant was spotted onto the MALDI target plate covered by 1 µl of HCCA matrix and used for mass spectrometry analysis. Peaks were manually selected using the flexAnalysis 3.4 software (Bruker Daltonik). Only peaks belonging to the corresponding antibiotic drug and the respective degradation products were labeled.

**Results:** In the case of meropenem and imipenem, several peaks were present in spectra obtained from sensitive and resistant strains; however, it was not possible to distinguish characteristic profiles. On the contrary, the pattern observed for ertapenem allowed for the differentiation of sensitive and resistant strains. Carbapenem-sensitive strain revealed 498.7 Da molecular peak corresponding to the single sodium adduct of ertapenem. Furthermore, several adduct peaks were found, i.e., 514.7 Da, 520.7 Da, and 536.6 Da, representing [M+K]⁺, [M+2Na]⁺, and [M+Na+K]⁺ ertapenem derivatives, respectively. In the spectra of carbapenem-resistant strains, the presence of a 450 Da peak was found, which corresponds to the hydrolysed, decarboxylated form of ertapenem. The obtained differentiation of ertapenem-resistant and sensitive strains was congruent with MIC values. MALDI-TOF MS has shown its ability to correctly identify resistance to ertapenem in 30 min.

**Conclusion:** The most reliable results were obtained for ertapenem i.e., it was possible to clearly distinguish between susceptible and resistant profiles. The method is promising, despite the fact that there are some difficulties in the interpretation of the spectra caused by the formation of sodium and potassium adducts as well as cluster ions of the tested molecules.

**Acknowledgments:** The study was funded by statutory task S/446 The use of MALDI-TOF to identify bacterial resistance to selected classes of antibiotics. The Department of Omic Analyses is supported by the Ministry of Science and Higher Education grant (SPUB D/03/1.0).

<footnote n="1">¹Department of Omic Analyses, National Veterinary Research Institute, Pulawy, Poland</footnote>

<footnote n="2">²Department of Microbiology, National Veterinary Research Institute, Pulawy, Poland</footnote>
**The respiratory microbiome of piglets displays temporal development and harbours strains with putative probiotic use against common respiratory pathogens.**

A.A. Vlasblom<fnref n="1" />*, S. Patel<fnref n="2" />, C. Huseyin<fnref n="2" /> M. Claesson<fnref n="2" />, D. Crespo-Piazuelo<fnref n="4" />, L. Wadja<fnref n="4" />, P.G. Lawlor<fnref n="4" />, R.E.C. Luiken<fnref n="1" />, J.A. Wagenaar<fnref n="1" />,<fnref n="3" />, B. Duim<fnref n="1" />, and A.L. Zomer<fnref n="1" />

**Introduction** Bacterial pathogens residing in the porcine respiratory tract undermine animal health and welfare, and some are a zoonotic risk. We investigated the development of the porcine nasal microbiome to identify and isolate bacterial species negatively associated with common porcine respiratory pathogens. These isolated species have potential for *in vivo* pathogen reduction strategies.

**Methods** Nasal swabs were obtained from 252 piglets, 7 per litter from 36 sows, from 9 farms in 3 countries (Germany, Ireland, the Netherlands), at 16 sampling moments from birth till week 10 (n = 4032). DNA was isolated and duplicate samples were stored at -80C for bacterial isolation. Using V3-V4 16S rRNA (n = 813) and *tuf* (n = 538) gene sequencing data of overlapping samples, correlation analysis was performed to identify microbial associations using SparCC. Additionally, sequencing data was supplemented with *Staphylococcus aureus* specific qPCR data. Species negatively associated with *S. aureus* were identified using mixed model and rmcorr. They were isolated, followed by *in vitro* (phenotypical antimicrobial resistance testing) and *in silico* (whole genome sequencing, taxonomy, antimicrobial resistance genes, virulence factors) safety screening, following EFSA guidance.

**Results** Amplicon sequencing allowed us to describe the developing piglet nasal microbiome. From the microbiome data, network analysis identified 26 unique taxa negatively associated with pathogens as *Bordetella sp.*, *Glaesserella parasuis*, *Mycoplasma hyorhinis*, *Pasteurella multocida*, *S. aureus*, and *Streptococcus suis*. The found taxa' negative association was consistent over the three countries. qPCR supplementation to amplicon sequencing data identified a further 54 species negatively associated with *S. aureus*. Literature screening of the identified species/taxa resulted in 15 probiotic candidates against *S. aureus*. The candidates were consisting of lactic acid bacteria (LAB) and species closely related to the *Staphylococcus* genus. An isolation effort, followed by *in vitro* and *in silico* safety and efficacy studies, yielded three suitable LAB strains, meeting the Qualified Presumption of Safety (QPS) status from EFSA. These species were subsequently utilized in an animal trial.

**Conclusion** The porcine nasal microbiome displays a distinct development in time. This development is varying between individual piglets but on average consistent between the studied countries. Further investigation of the developing porcine nasal microbiome resulted in probiotic candidates aimed at pathogen reduction strategies. Three probiotic LAB strains against *S. aureus* are currently under study *in vivo*.

<footnote n="1">¹Division of Infectious Diseases and Immunology, Faculty of Veterinary Medicine, Utrecht University, the Netherlands.</footnote>

<footnote n="2">²APC Microbiome, University College Cork, Cork, Ireland.</footnote>

<footnote n="3">³Wageningen Bioveterinary Research, Lelystad, the Netherlands.</footnote>

<footnote n="4">⁴Pig Development Department, Animal & Grassland Research and Innovation Centre Teagasc, Moorepark, Ireland</footnote>
# Does hyperpersistence influence the evolution of *Escherichia coli* toward fluoroquinolone resistance ?

Aurore Perault, Quentin VALLÉ, Marlène LACROIX, Alicia HUESCA, Alain BOUSQUET-MELOU and Etienne GIRAUD

UMR 1436 INRA/ENVT INTHERES, Toulouse, France

Persistence is a phenomenon in which a subpopulation of bacteria, known as "persisters," enters a dormant state, enabling them to transiently survive bactericidal antibiotic concentrations. Hyperpersistent strains produce a much higher proportion of persisters in their populations, which can compromise infection treatments. Furthermore, previous studies have shown that hyperpersistence can promote the acquisition of "classical" antibiotic resistance, i.e., the ability of bacteria to grow in the presence of antibiotics.

Previous studies on persisters were conducted using static antibiotic concentrations. However, in this study, we aimed to test whether hyperpersistence would promote the acquisition of fluoroquinolone resistance in *Escherichia coli* under fluctuating antibiotic concentrations that better mimic real-world therapy. To achieve this, bacteria were grown in a dynamic in vitro system where they received fresh medium continuously, and ciprofloxacin twice a day to reach a peak concentration of 1 µg/mL. We examined the evolution of populations of three isogenic *E. coli* strains that displayed either a susceptible phenotype (S), hyperpersistence (H, *gyrB* L422P mutation), or low-level resistance to fluoroquinolones (R, *gyrB* S464Y mutation), under exposure to fluctuating ciprofloxacin concentrations over a 10-day period. We monitored bacterial populations as well as their level of susceptibility to ciprofloxacin during the experiment.

Regardless of their initial phenotypes, the three *E. coli* strains showed adaptation of their populations, allowing them to better survive the repeated ciprofloxacin doses over the 10-day treatment. Similar levels of resistance to ciprofloxacin and survival rates were observed in the bacterial populations and clones derived from the three parental strains. Additional *gyrA* quinolone resistance mutations were identified in clones derived from the S and R parental strains, but not from the hyperpersistent strain.

In conclusion, in our experimental conditions, hyperpersistence did not appear to promote the evolution of bacteria toward high ciprofloxacin resistance levels, but may have influenced the mechanism of adaptation to the 10-days treatment.

## References

[1] Perault A, Turlan C, Eynard N, Vallé Q, Bousquet-Mélou A, Giraud E. *Front Microbiol.* 13:908296
# Horizontal gene transfer between bacteria inside the intestinal microbiota of the nematode *Caenorhabditis elegans*

Romane Dusfour-Castan, Phan Thai Nguyen Le, Patricia Siguier and Jérôme Rech, Bao Ton-Hoang and Catherine Guynet

*Laboratoire de Microbiologie et de Génétique Moléculaires, Centre de Biologie Intégrative (CBI), Centre National de la Recherche Scientifique, Université de Toulouse, UPS, F-31000, Toulouse, France.*

Bacteria have the remarkable property of being able to adapt quickly and continuously to their environment, thanks to their ability to exchange and combine mobile genetic elements (MGE). These MGEs, which notably include conjugative plasmids and transposons, are the main carriers of antibiotic resistance genes. They thus promote the emergence of multi-resistant bacteria, a serious threat currently weighing on global health.

So far, little is known about the propagation of these MGEs within complex bacterial communities. Here, we aim to study the spatiotemporal dynamics of MGE exchanges using the nematode *Caenorhabditis elegans* as a simple experimental model of a gastrointestinal system harboring controlled bacterial communities. The natural microbiota of *C. elegans* has only recently been described, opening up the possibility of using this simple yet powerful model system in studies of the host microbiota (Zhang et al., 2017). The composition of the microbiota, which is composed of a high diversity of bacterial species, the dominant group comprising several Y-proteobacteria (*Enterobacteriaceae*, *Pseudomonaceae* and *Xanthomonodaceae*), was shown to tune *C. elegans* health (Haçariz et al., 2021) and pathogenesis (Radeke and Herman, 2021). *C. elegans* is also a model of choice for our study for technical reasons, such as ease of culture, short life cycle, selfing hermaphroditism allowing maintenance of homozygous cultures, the possibility to generate large numbers of nearly genetically identical individuals with identical life histories, and importantly transparency under light microscopy allowing direct visualization of bacterial cells *in vivo*. We use the CeMBio bacteria as a simplified natural *C. elegans* microbiota (Dirksen et al., 2020). This includes a dozen of environmental bacterial strains from several classes (α-, β- and γ-proteobacteria and Bacteroidetes) that have been selected from meta-analyses of the natural microbiome of the nematode.

We first focus on the establishment of the CeMBio community inside *C.elegans'* intestine, which is poorly described (i.e. localization, proportion and stability along time of the different bacterial species of the microbiota in the intestine). We are also constructing the tools that allow us to analyze and compare the fate of several broad host range conjugative plasmids and transposons from several families within the CeMBio community, both *in vitro* on agar plates or in liquid cultures and *in vivo*, in the gut of *C. elegans* intestine.

By combining several approaches including bacterial genetics, fluorescence microscopy, and bioinformatics, our data could provide a better understanding of how MGEs propagate in complex ecosystems such as microbiota and thus contribute to the research for ways to limit the emergence of multi-resistant bacteria.
# Antimicrobial residues and the minimal selective concentration

A.F. Swinkels<fnref n="1" />*, E.A.J. Fischer<fnref n="1" />, B.J.A. Berendsen<fnref n="3" />, J.A. Wagenaar<fnref n="1" />,<fnref n="2" />, A.L. Zomer<fnref n="1" />

## Introduction

The global emergence of antimicrobial resistance (AMR) is a cause for concern. Use of antimicrobials contributes to AMR. A particular problem in the use of antimicrobials in livestock is that antimicrobial residues are excreted in the same environment in which the animals are kept. It is believed that after the application of antimicrobials, they are partly excreted through urine and feces and thereafter quickly degrade. However, some antimicrobials proved to be very persistent. In this study, we examined whether persistent antimicrobial compounds remain longer in the animal environment and if the remaining concentrations still have selective properties by determining the minimal selective concentration (MSC).

## Methods

The MSC was determined through fecal fermentation studies. An animal trial was conducted to determine the persistence of the three antimicrobials over time. Four groups of broilers were divided in three subgroups (n=12). Groups were left untreated (control) or were treated with amoxicillin (non-persistent), doxycycline or enrofloxacin (persistent). We determined the resistome by shotgun metagenomics using Illumina sequencing and did an phenotypical resistance analysis of *Escherichia coli* isolates. Antimicrobials were extracted from the fecal samples and analysed by LC-MS/MS.

## Results

After treatment, persistent antimicrobials (doxycycline and enrofloxacin) had concentrations equal to or higher than the MSC established in the fermentation study, in contrast to the amoxicillin treatment. The doxycycline treatment showed an increase in phenotypically resistance and an increase in resistance genes in the resistome. In contrast, the amoxicillin treatment only showed an increase in the resistance genes directly after treatment. Enrofloxacin treatment resulted in merely enrofloxacin resistant *E. coli* and the resistance genes in the resistome increased during the experiment.

## Conclusions

Persistent antimicrobials remain longer in the animal environment at selective concentrations. Our findings suggest that persistency of antimicrobials should be taken into consideration in the assessment of priority classification of antimicrobials.

<footnote n="1">¹Faculty of Veterinary Medicine, Utrecht University, the Netherlands.</footnote>

<footnote n="2">²Wageningen Bioveterinary Research, Lelystad, the Netherlands.</footnote>

<footnote n="3">³Wageningen Food Safety Research, Wageningen, the Netherlands</footnote>
<figure><img src="image_61.png" /></figure>

Development of first quality control ranges for biocide susceptibility testing

Angela R. SCHUG<sup>1,2</sup>, Anissa D. SCHOLTZEK<sup>1,2</sup>, John TURNIDGE<sup>3</sup>, Marita MEURER<sup>4,5</sup>, Stefan SCHWARZ<sup>1,2</sup>, Andrea T. FEßLER<sup>1,2</sup>

**Introduction:** Quality control (QC) is important for laboratory tests. Therefore, the aim of this study was to develop QC ranges for biocide susceptibility testing (BST).

**Materials and Methods:** Four reference strains, namely *Staphylococcus aureus* ATCC® 6538, *Enterococcus hirae* ATCC® 10541, *Escherichia coli* ATCC® 10536 and *Pseudomonas aeruginosa* ATCC® 15442, were chosen as quality control strains. During an interlaboratory trial eleven laboratories tested these four strains and four biocides, namely benzalkonium chloride, chlorhexidine, octenidine and polyhexanide ten times using broth microdilution with three lots of tryptic soy broth. The RangeFinder software was used for data analysis.

**Results:** Overall, comparable results were observed by the eleven laboratories. Identified outliers (up to seven) were excluded from the QC range establishment. New QC ranges were established for all but two reference strain-biocide combinations and comprise three to five dilution steps (Table 1). Due to the limited solubility of the biocides in the test range required for *P. aeruginosa* ATCC® 15442, QC ranges for chlorhexidine and polyhexanide could not be determined for this reference strain. Therefore, we recommend using *Escherichia coli* ATCC® 10536 as reference strain in parallel with *Pseudomonas* field isolates, when testing chlorhexidine and polyhexanide [1].

<table><caption>Table 1: Summary of the new quality control ranges (minimal inhibitory concentrations in %)</caption><thead><tr><th>Biocide</th><th>S. aureus<br>ATCC® 6538</th><th>E. hirae<br>ATCC® 10541</th><th>E. coli<br>ATCC® 10536</th><th>P. aeruginosa<br>ATCC® 15442</th></tr></thead><tbody><tr><td>Benzalkonium chloride</td><td>0.00003-0.00025</td><td>0.000125-0.0005</td><td>0.0005-0.002</td><td>0.002-0.008</td></tr><tr><td>Chlorhexidine</td><td>0.00003-0.00025</td><td>0.00003-0.00025</td><td>0.000015-0.00025</td><td>-</td></tr><tr><td>Polyhexanide</td><td>0.00006-0.001</td><td>0.000125-0.002</td><td>0.00006-0.001</td><td>-</td></tr><tr><td>Octenidine</td><td>0.00006-0.00025</td><td>0.00006-0.0005</td><td>0.00006-0.0005</td><td>0.000125-0.002</td></tr></tbody></table>

**Conclusions:** After developing harmonized BST methods [2], this study was the subsequent step. Together, these studies will contribute to the harmonisation and validation of the BST in the future.

**References**

[1] Schug AR Scholtzek AD, Turnidge J, Meurer M, Schwarz S, Feßler AT and The Biocide Susceptibility Study Group. (2022). *Pathogens*. 11(2):223.

[2] Schug AR Bartel A, Scholtzek AD, Meurer M, Brombach J, Hensel V, Fanning S, Schwarz S, Feßler AT. (2020). *Vet Microbiol.* 248:108791.

<footnote n="1"><sup>1</sup>Institute of Microbiology and Epizootics, Centre for Infection Medicine, School of Veterinary Medicine, Freie Universität Berlin, Berlin, Germany</footnote>

<footnote n="2"><sup>2</sup>Veterinary Centre for Resistance Research (TZR), School of Veterinary Medicine, Freie Universität Berlin, Berlin, Germany</footnote>

<footnote n="3"><sup>3</sup>School of Biological Sciences, University of Adelaide, Adelaide, Australia</footnote>

<footnote n="4"><sup>4</sup>Institute for Biochemistry; University of Veterinary Medicine Hannover, Hannover, Germany</footnote>

<footnote n="5"><sup>5</sup>Research Center for Emerging Infections and Zoonoses (RIZ); University of Veterinary Medicine Hannover, Hannover, Germany</footnote>
Antibiotic resistance in Australian leafy greens pre-farm gate

Sabina Shrestha<fnref n="1" />, Janine Muller<fnref n="1" />, Yuhong Liu<fnref n="1" />, Rachel Hodgeman<fnref n="1" />,
Rachel Mann<fnref n="2" />, Brendan Rodoni<fnref n="1" /> and Fiona Constable<fnref n="1" />

Fresh produce, including leafy greens are promoted as being a healthy source of vitamins and minerals, and are consumed daily worldwide. The increased consumption of fresh horticultural produce has been closely followed by an increase in foodborne disease outbreaks. Outbreaks caused by fresh horticultural produce has overtaken those caused by meat, dairy and seafood. This pathway can be a silent source of introduction and exposure for consumers, to antimicrobial residues, antimicrobial-resistant (AMR) bacteria and antimicrobial resistance genes (ARGs) of clinical importance. Exposure to AMR bacteria through the gastrointestinal tract may lead to drug resistant infections or the potential for the sharing of ARGs with other gut microflora eventually decreasing the effect of medicine and making infections harder to treat. So, the question is: are our leafy greens making us stronger and healthier or strengthening bacteria against antimicrobial treatment?

The major factors that contribute to the emergence and spread of AMR in leafy greens involve the use of antibiotics in plant production and livestock farming, as well as use of contaminated irrigation water, manure or soil. Moreover, the microbiomes of plants and soil that are involved in nutrient cycling, plant growth, and disease resistance, might also be acting as hotspots for the development and spread of AMR. Understanding the dynamics of microbial communities and their AMR profiles in horticultural systems is necessary to make informed strategies for reducing the risk of AMR. Knowledge of the presence and prevalence of AMR in the Australian horticulture sector is limited. So, the major aim of this project is to characterise the diversity of AMR genes and their distribution in commensal, plant pathogenic and food safety bacteria in the Victorian horticultural sector during pre-farm gate production and processing.

Metagenomic sequencing will be used to study the microbiome of leafy greens and associated antibiotic resistance genes (ARGs). The genes and mobile genetic elements will be identified, in both microbiome and from select bacterial isolates, to determine ARG prevalence in leafy greens. The AMR susceptibility of the select bacterial isolates will be confirmed to determine whether the ARGs are expressed. This PhD project will focus on:

1. The development of workflows to extract DNA from leafy greens for metagenomic sequencing

2. The development of bioinformatic pipelines to characterise the microbiome and associated ARGs

3. Studying the genetic links of ARGs and mobile genetic elements in select bacterial isolates to determine the possibility of horizontal gene transfer of ARGs in horticulture sector prefarm gate.

We are hopeful that this genotypic approach supported by phenotypic testing can provide important insights into the prevalence and diversity of ARGs in Australian horticulture pre-farm gate and help to mitigate the risks of AMR from farm-to-fork.

We are hopeful that this genotypic approach supported by phenotypic testing can provide important insights into the prevalence and diversity of ARGs in Australian horticulture pre-farm gate and help to mitigate the risks of AMR from farm-to-fork.

<footnote n="1">¹Agiculture Victoria, Department of Energy, Environment and Climate Action (DEECA), Victoria, Australia</footnote>

<footnote n="2">²Biosecurity & Agriculture, Department of Energy, Environment and Climate Action (DEECA), Victoria, Australia</footnote>
# Pyocin S2 Conjugates for the Treatment of PyoS2-Resistant MDR Clinical Isolates of *Pseudomonas aeruginosa*.

Tim Moritz WEBER<fnref n="1" />, Colin MacKENZIE<fnref n="2" />, and Jörg PIETRUSZKA<fnref n="1" />,<fnref n="3" />

The opportunistic human pathogen *Pseudomonas aeruginosa* is a Gram-negative bacterium that can be isolated from environmental sources, but is of great concern when found in a clinical context.[1] Widespread multidrug resistance (MDR) mechanisms against common antibiotics, reserve group antibiotics, and environmental toxins make *P. aeruginosa* difficult to treat and prompted the World Health Organization (WHO) in 2017 to list *P. aeruginosa* with priority 1 (“critical”) on a list of pathogens, for which new antibiotics or promising alternatives are urgently needed.[2] An often mentioned potential alternative are so-called bacteriocins; however, bacteriocins of Gram-negative bacteria are lacking proof of applicability so far.[3] Bacteriocins are toxic multi-domain proteins and are found in most bacterial Gram-negative and Gram-positive species. Other than most small molecule antibiotics, bacteriocins are narrow band antibiotics, mostly acting on closely related strains of the same genus and are evolutionary relevant for the fight for ecological niches. Selectivity and host specificity rely on a conserved uptake mechanism, which involves the outer membrane protein machinery of the bacterial target cell. To date, several bacteriocins have been found in *P. aeruginosa* □ called pyocins □ that kill other strains of *P. aeruginosa*. The producing strain itself is protected from the pore-forming or nuclease killing activity of pyocins by co-expression of immunity proteins that confer resistance to this particular strain.[4] Here we present preliminary proof that resistance against the DNase pyocin S2 (PyoS2) can be overcome by partial blockage of the interaction site of the PyoS2 cytotoxic domain and its cognate immunity protein ImS2. *In vitro* labeled toxin with a non-toxic fluorophore label was shown to kill the PyoS2-producing *P. aeruginosa* PAO1 and several PyoS2-resistant MDR clinical isolates of *P. aeruginosa*. We believe that our findings might contribute to bringing bacteriocins of Gram-negative strains closer to application as potential substitutes for antibiotics or as extension of our common antibiotics portfolio for treatment of clinically relevant MDR strains in the future.

<figure><img src="image_62.png" /><figcaption>**Fig. 1:** Schematic depiction of the translocation mechanism of pyocin S2 and its conjugates across the bacterial membrane of *Pseudomonas aeruginosa*. The outer membrane porin FpvAII serves as primary receptor and is hijacked by PyoS2 derivatives, the inner membrane TonB machinery energizes the import into the periplasm. Protease-assisted cleavage of pyoS2 leads to translocation into the cytoplasm, where the C-terminal cytotoxic domain of PyoS2 (DNase activity) causes cell death by degradation of genomic information.</figcaption></figure>

## References

[1] Wolfgang, M. C. et al., *PNAS* **2003**, *100*(14), 8484-8489.

[2] https://www.who.int/news/item/27-02-2017-who-publishes-list-of-bacteria-for-which-new-antibiotics-are-urgently-needed

[3] Chikindas, M. L. et al., Curr. Opin. Biotechnol. **2018**, *49*, 23-28.

[4] Michel-Briand, Y., & Baysse, C., *Biochimie* **2002**, *84*(5-6), 499-510.

<footnote n="1">¹Institute of Bioorganic Chemistry (IBOC), Heinrich Heine University, Düsseldorf, Germany</footnote>

<footnote n="2">²Institute of Medical Microbiology and Hospital Hygiene, University Clinics Düsseldorf (UKD), Düsseldorf, Germany</footnote>

<footnote n="3">³Institute for Bio- and Geosciences (IBG-1), Research Center Jülich, Jülich, Germany</footnote>
# Impact of minocycline, an “old” antibiotic, on the digestive flora in pigs.
## A study model for humans.

Dupouy V., Roques B. B., Lacroix M. Z., Vallé Q., Arpaillange N., Bibbal D., Ramon-Portugal F., Imazaki P., Ferran A. A., Bousquet-Mélou A.

INTHERES, Université de Toulouse, INRAE, ENVT, Toulouse, France

**Introduction:** The increase of multidrug-resistant (MDR) pathogens has renewed interest in old antibiotics. The combination of polymyxin B and minocycline for intravenous antibiotic therapy in human is promising. The drawback is the elimination of minocycline in the intestines, with potential impact on the digestive microbiota. Our objectives were to 1) determine the pharmacokinetic parameters of minocycline in pigs to mimic the expected exposure in humans and 2) assess the impact of minocycline administration on the intestinal microbiota.

**Materials and Methods:** Pigs received saline (Control group) or minocycline by intravenous route at a low dose (LD group, 8 mg/Kg/d for 4 days) or at high dose (HD group, 16 mg/Kg/d for 4 days). For the determination of pharmacokinetic parameters in the pharmacokinetic study (n=8 per group), minocycline concentrations in plasma were determined by U-HPLC with UV detection. For fecal microbiome evaluation, antibiotic resistance was evaluated by cultural methods and quantitative real-time PCR. The impact on microbial diversity was evaluated by sequencing the V3-V4 region of the 16S rRNA gene.

**Results:** Pharmacokinetic data indicated that minocycline was eliminated with a clearance 3 to 7 times higher in pigs compared to humans. Thus, in order to obtain exposure of pigs similar to that of humans, 8 to 16 mg/Kg/d were administered to pigs. High dose minocycline (16 mg/Kg/d) increased the proportion of minocycline-resistant *Enterobacterales* (-2.5Log to -0.5Log) after 4 days of treatment while the total (~10⁵ UFC/g faeces) and minocycline-resistant *Enterobacterales* (10²-10³ UFC/g faeces) were unchanged. Quantification of the 95 genes coding for antibiotic resistance (minocycline, tetracycline, other antibiotic families) or genes linked to genetic mobile elements (transposon, insertion sequence, integron) showed the increase of the relative abundance of the *tet*(G) gene. No impact was detected on other genes. The majority of 16S rRNA sequences belonged to *Firmicutes* and *Bacteroides*. The beta-diversity index and nonmetric multidimensional scaling demonstrated changes in microbial membership after 4 days of minocycline treatment. Ninety-eight OTU were differentially abundant. However, bacterial phylotypes did not shifted with minocycline treatment showing similar rate of *Firminutes* and *Bacteroides* in treated or control pigs.

**Conclusion:** High dose minocycline treatment seems to have a weak impact on the digestive microbiome (resistance and diversity).

**Funding:** JPIAMR and ANR under the research grant CO-ACTION.
# GenoFig: a user-friendly application for vizualisation and comparison of genomic regions.

Maxime BRANGER and Sébastien LECLERCQ

INRAE, Université de Tours, UMR ISP, F-37380, Nouzilly, France

Most concerns about antibiotic resistance are linked to the extremely efficient dissemination of mobile genetic elements carrying resistance genes, such as plasmids and conjugative transposons. Understanding the evolution of these elements usually requires visual comparison of replicons hosted by various bacterial strains or species. Here we present GenoFig, a graphical application for the generation of comparative genomics figures, intended to be as easy to use as possible for biologists and flexible enough to adapt to a variety of needs. GenoFig allows the personalized representation of annotations extracted from GenBank files in a consistent way across sequences, using regular expressions. It also provides several unique options to optimize the display of homologous regions between sequences, as well as other more classical features such as GC percent or GC-skew representations. In summary, GenoFig is a simple, free, and highly configurable tool to explore the evolution of specific genomic regions and to produce publication-ready figures.

<figure><img src="image_63.png" /><figcaption>Example of figure produced with GenoFig</figcaption></figure>
# Quantification and molecular characterization of vancomycin resistant Enterococcus faecium (VRE) in hospital wastewater before, during and after a hospital outbreak

Hetty BLAAK<fnref n="1" />, Djamayl ENGELEN<fnref n="2" />, Merel KEMPER<fnref n="1" />, Jean-Luc MURK<fnref n="2" />, Anne MUTSAERS-VAN OUDHEUSDEN<fnref n="2" />, Ana Maria DE RODA HUSMAN, Heike SCHMITT<fnref n="1" /> and Joep STOHR<fnref n="2" />

**Background and Objectives.** With the COVID-19 pandemic, sewage-based surveillance as a monitoring tool of rare micro-organisms in the human population has gained more interest globally. Sewage-based surveillance is typically performed at the municipal wastewater treatment plant level to cover large populations. The current study aimed to investigate sewage-based surveillance at an institutional level as a tool to support the detection and management of hospital outbreaks and infection prevention and control, focusing on vancomycin-resistant *E. faecium* (VRE).

**Methods.** Wastewater was sampled from a large Dutch teaching hospital, between March 2022 and April 2023, using an automated sampler at a baseline frequency of once per month. Between 14 November 2022 and 6 March 2023 the hospital was confronted with a VRE outbreak involving 18 patients at three wards. Because of this, the sampling frequency was increased to once weekly as of January 2023. Wastewater samples were membrane filtered, followed by enrichment in vancomycin-supplemented broth and subsequent plating on selective agar. Initially, a semi-quantitative detection method was used. As of September 2022, the numbers of VRE were determined using a most probable number method. Suspected VRE colonies were confirmed using MALDI-TOF, E-test and vancomycin-PCR. From the start of the outbreak all hospital patients at affected wards were screened weekly for the presence of VRE, and a contact investigation was done after finding a new case. Whole genomes of VRE isolates from patients and wastewater were sequenced using Illumina Miseq and compared using cgMLST analysis using a cluster distance of 20 nucleotides. VRE concentrations and cgMLST variants in wastewater were analysed relative to the presence of VRE-positive patients before, during and after the outbreak.

**Results.** VRE was detected in all 20 wastewater samples. Before the outbreak, four different unrelated sequence variants were identified at different time-points, among which the outbreak variant (3/10 isolates). From two months before the outbreak till the end of the outbreak, VRE concentrations ranged between 10⁵ - 10⁶ MPN per litre and only the outbreak variant was detected (12/12 isolates). After that, VRE concentrations declined quickly to 10³ MPN per litre and variant variability was observed again, consisting of the outbreak variant (1/3 isolates) and one of the pre-outbreak variants. These results are preliminary, as not all sequence data were available at the time of writing.

**Conclusion.** High VRE concentrations in wastewater preceded the identification of the hospital outbreak in patients, and concentrations subsequently followed the outbreak dynamics. During the outbreak, VRE sequence types of the isolates shifted from multiple unrelated types to solely the outbreak-related type. Although further investigation is required, these aspects suggest that wastewater surveillance may be a valuable tool to support the management of hospital outbreaks.

<footnote n="1">¹RIVM, Bilthoven, The Netherlands</footnote>

<footnote n="2">²Elisabeth TweeSteden Ziekenhuis, Tilburg, The Netherlands</footnote>
<figure><img src="image_64.png" /></figure>

ABRomics: An integrated multi-omics platform for antibiotic resistance research and public health

Julie LAO<sup>1,3</sup>, Pierre MARIN<sup>1,2</sup>, Romain DALLET<sup>1,4</sup>, Fabien MAREUIL<sup>7</sup>, Alix DE TOISY<sup>7</sup>, Kenzo-Hugo HILLION<sup>5</sup>, Aurélien BIRER<sup>6</sup>, Nadia GOUE<sup>2</sup>, Richard BONNET<sup>6</sup>, Etienne RUPPE<sup>3</sup>, Gildas LE CORGUILLE<sup>1,4</sup>, Philippe GLASER<sup>8</sup>, Claudine MEDIGUE<sup>1,9</sup>

<sup>1</sup>CNRS, Institut Français de Bioinformatique, IFB-core, UAR 3601 - Évry (France)

<sup>2</sup>AuBi platform, Mésocentre, Université Clermont-Auvergne - Aubière (France)

<sup>3</sup>Université Paris Cité and Université Sorbonne Paris Nord, Inserm, IAME - Paris (France)

<sup>4</sup>Sorbonne Université, CNRS, FR2424, ABiMS, Station Biologique - Roscoff (France)

<sup>5</sup>Faculté des Sciences Techniques, Université de Nantes, F-44000 - Nantes (France)

<sup>6</sup>CHRU Clermont-Ferrand Gabriel-Montpied - Clermont-Ferrand (France)

<sup>7</sup>Institut Pasteur, Université Paris Cité, Bioinformatics and Biostatistics Hub, F-75015 - Paris (France)

<sup>8</sup>Institut Pasteur, Unité EERA, CNRS UMR604, F-75015 - Paris (France)

<sup>9</sup>CNRS UMR8030, Université Evry-Val-d'Essonne, CEA, Genoscope, LABGeM - Évry (France)

Antibiotic resistance (ABR) is a major public health issue prioritized for mitigation by international institutions. Multidrug resistant bacteria (MDRB) and Antibiotic Resistance Genes (ARGs) carried by mobile genetic elements spread between the human, animal, and environmental sectors. Whole Genome Sequencing (WGS) is used for molecular typing purposes at the highest resolution. It provides identification of ARGs and their genetic supports as well as mutations leading to a decrease in antibiotic susceptibility. Epidemiological and WGS data are used for tracking MDRB in hospital outbreaks but also across the animal and environmental sectors. Sharing and interoperability of high-quality data (sequence and metadata) are key requirements for addressing the spatio-temporal dissemination of MDRB. To this aim, the French Priority Plan on ABR has funded the development of an online, open platform dedicated to antibiotic resistance.

We are establishing a repository of structured, interoperable, standardized, and well-annotated multi-omics data with tailored mathematical and bioinformatics tools to answer generic and specific research questions related to ABR. The ABRomics platform includes standardized pipelines to run ABR analyses of WGS from pathogenic strains supported with integrated databases (ARG, sequence types [ST], virulence factors [VF]). Uploading data, launching pipelines, viewing and cross-referencing enriched results will be achieved through easy-to-use web interfaces. ABRomics β-version integrating the ABR detection genomic pipeline and other markers such as ST, and VF will be available to the consortium in summer 2023 and to the whole microbial research community by the end of 2023. Core-genome multi-locus sequence typing, relationships between strains and metagenomics pipelines will next be made available.

**Acknowledgement of grants and fundings:**

This work is financially supported by the French Priority Research Programme on Antimicrobial Resistance (PPR antibioresistance), coordinated by Inserm and funded by the Secretaire General Pour L'investissement (SGFI)
Session 5 - Novel approaches, methods and tools dedicated to antimicrobial
resistance (detection, evolution, diagnostics, surveillance)

P85

Phenotypic and WGS-based characterization of antimicrobial resistance of
*Trueperella pyogenes* clinical isolates from humans and animals in Switzerland

Emma MARCHIONATTI<fnref n="1" />,<fnref n="2" />, Sonja KITTL<fnref n="2" />, Parham SENDI<fnref n="3" />, Vincent PERRETON<fnref n="2" />

*Trueperella pyogenes*, a commensal Gram-positive facultative-anaerobic rod of the skin and mucous membranes of animals can cause suppurative infections in animals, and rarely humans (1). These infections are commonly treated with antibiotics, posing the risk of selecting resistant bacteria through the acquisition of mobile genetic elements containing antimicrobial resistance genes (2). The objectives of the present study were to determine i) the phenotypic and genotypic antimicrobial resistance profiles of *T. pyogenes* in Switzerland, and ii) the genetic relatedness between strains of different origins.

Thirty-five *T. pyogenes* isolates from animals and 8 from humans were obtained from a veterinary and a human diagnostic facility in Switzerland. The strains were cultivated on TSA-S (Becton, Dickson) under 5% CO₂ atmosphere at 37°C for 24 hours. Identification was confirmed by MALDI-TOF mass spectrometry (Bruker). Minimal inhibitory concentrations (MICs) of 19 antibiotics and 3 antiseptics were determined by broth microdilution method following CLSI recommendations. The MICs were interpreted using breakpoints derived from previously published data. Genomic DNA was extracted using Masterpure™ purification kit (Lucigen) and sequenced at the NGS Platform, University of Bern, Switzerland, using PacBio HiFi technology. Reads were demultiplexed and *de novo* assembled using Flye v.2.9.1. The 43 complete genomes were screened *in silico* for antimicrobial resistance genes using Resfinder v.4.1 (Center for Genomic Epidemiology) and CARD-RGI (McMaster University). Core genome-based MLST (cgMLST) analysis was performed for phylogenetic relatedness.

Seventeen isolates were susceptible to all tested antibiotics. Resistant test results to tetracycline (n=23), streptomycin (n=20), and sulfonamides (n=17) were common in both animal and human isolates, while resistance to erythromycin (n=4), clindamycin (n=3), trimethoprim (n=2), and chloramphenicol (n=2) was only sporadic and predominantly detected in animal isolates. The resistance genes *tet*(W), *aad*A9, and *sul1* were the most frequently detected, reflecting the phenotypic resistance profile. Phenotypic resistance to quaternary ammonium compounds was not observed despite the frequent detection of the resistance gene *qacE*Δ1. Two large clusters were identified by cgMLST, revealing that human isolates were more closely related to those from cattle compared to other animal species.

The presence of genetically related *T. pyogenes* strains in humans and animals suggests that exchange of mobile genetic elements may occur. Acquired resistances to several antimicrobials indicates that judicious use of antibiotics should be made based on antimicrobial susceptibility testing.

**References**

[1] Rzewuska M, Kwiecien E, Chrobak-Chmiel D, Kizerwetter-Swida M, Stefanska I, Gierynska M. 2019. Pathogenicity and virulence of *Trueperella pyogenes*: a review. *Int J Mol Sci* 20(11):2737

[2] Feßler AT, Schwarz S. 2017. Antimicrobial resistance in *Corynebacterium* spp., *Arcanobacterium* spp., and *Trueperella pyogenes*. *Microbiol Spectr* 5(6) DOI: 10.1128/microbiolspec.ARBA-0021-2017

<footnote n="1">¹Department of Clinical Veterinary Science, Vetsuisse Faculty, University of Bern, Switzerland</footnote>

<footnote n="2">²Institute of Veterinary Bacteriology, Vetsuisse Faculty, University of Bern, Switzerland</footnote>

<footnote n="3">³Institute for Infectious Diseases, Faculty of Medicine, University of Bern, Switzerland</footnote>
# Antimicrobial resistance trends of *Salmonella* spp. isolated from yellow-legged gulls (*Larus michahellis*) from Medes Islands (Catalonia, NE Spain)

Aniol MARULL<fnref n="1" />,<fnref n="2" />, Teresa AYATS<fnref n="1" />,<fnref n="2" />, Raül RAMOS<fnref n="3" />,<fnref n="4" />, Jacob GONZÁLEZ-SOLÍS<fnref n="3" />,<fnref n="4" />, and Marta CERDÀ-CUÉLLAR<fnref n="1" />,<fnref n="2" />

Salmonellosis is the second most commonly reported foodborne gastrointestinal infection in humans in the EU. Not only food animals are carriers of *Salmonella*, but also wildlife. These particularly include many gull species that scavenge on refuse and can act as *Salmonella* reservoirs, including their antimicrobial resistant (AMR) strains. Our former study already showed the role of yellow-legged gulls (*Larus michahellis*) in the epidemiology of *Salmonella* and their AMR in southern Europe. Here, we have monitored one colony of this gull with a higher anthropogenic pressure in NE Spain, the Medes Islands, to assess its occurrence and trend of AMR *Salmonella*. We have focused on the most relevant *Salmonella* serovars responsible of human salmonellosis in the EU, that is, Enteritidis, Typhimurium and its monophasic variant. Assessment of the AMR of isolates (n= 38) recovered in 2013, 2017, 2018, 2020, 2021 and 2022 has been performed by means of a broth microdilution method using EUSVEC plates. Overall *Salmonella* spp occurrence over the years peaked at 80% in 2020 and bottomed out at 38% and 33%, in 2013 and 2021, respectively. Serovar Enteritidis was the less frequent, being isolated only in 2013. On the contrary, ser Typhimurium was isolated in 2013, 2017 and 2022, while its monophasic variant was the most frequent, and was isolated in all years. Overall, a high prevalence of AMR was found, with 95% of tested isolates showing resistance to at least one antimicrobial. Also, the prevalence of multidrug resistance (MDR) was high (55%), with monophasic *S. Typhimurium* showing the highest prevalence (63%), while *S. ser. Enteritidis* did not show MDR. However, a decreasing trend of MDR in the monophasic variant was observed across years (from 100% in 2013 to 33% in 2022). The most prevalent MDR profile was Ampicillin-Tetracycline-Sulfamethoxazole. However, there were isolates showing resistance up to 5 antimicrobial classes, although with a very low frequency. The three *S. ser. Enteritidis* showed resistance to quinolones (n=2) or to sulfamethoxazole alone (n=1). The decreasing trend of AMR shown by the monophasic variant of *S. Typhimurium* might be a result of the important reduction of antimicrobial use in both veterinary and human medicine during last years in Spain (and the EU in general). These findings highlight the importance of gulls in the epidemiology of AMR and support the potential of yellow-legged gulls as sentinels for environmental AMR in the Mediterranean region and the need to tackle this public health concern from a One Health approach.

<footnote n="1">¹Unitat mixta d'Investigació IRTA-UAB en Sanitat Animal. Centre de Recerca en Sanitat Animal (CReSA). Campus de la Universitat Autònoma de Barcelona (UAB), Bellaterra, 08193, Catalonia. Spain.</footnote>

<footnote n="2">²IRTA. Programa de Sanitat Animal. Centre de Recerca en Sanitat Animal (CReSA). Campus de la Universitat Autònoma de Barcelona (UAB), Bellaterra, 08193, Catalonia. Spain.</footnote>

<footnote n="3">³Institut de Recerca de la Biodiversitat (IRBio), Universitat de Barcelona, Av Diagonal 643, Barcelona 08028, Spain.</footnote>

<footnote n="4">⁴Departament de Biologia Evolutiva, Ecologia i Ciències Ambientals, Universitat de Barcelona, Av Diagonal 643, Barcelona 08028, Spain.</footnote>
# Gulls in southern Spain as carriers and potential spreaders of *Salmonella* spp. and antibiotic-resistant strains

<u>Mª Pilar GONZÁLEZ-NAVARRO</u><fnref n="1" />,<fnref n="2" />, Teresa AYATS<fnref n="1" />,<fnref n="2" />, Salvador GARCÍA-BARCELONA<fnref n="3" />, Jacob GONZÁLEZ-SOLÍS<fnref n="4" />, Marta CERDÀ-CUÉLLAR<fnref n="1" />,<fnref n="2" />

Some gull species, particularly those with scavenging feeding habits, can act as reservoirs of infectious agents and play an important role in their dissemination and maintenance in the environment [2]. This includes zoonotic agents such as *Salmonella*, which in turn can show resistance to antimicrobials of veterinary or human relevance. To gain insight into the epidemiology of *Salmonella* spp. and their antimicrobial-resistant strains, isolates recovered during 2022-2023 from two different large gull species in southern Spain were analysed. A total of 369 birds were sampled in urban or nearby urban areas: *Larus fuscus* (n=134) and *Larus michahellis* (n=235). To assess the presence of antibiotic-resistant (AMR) strains, the isolates were characterized phenotypically using a broth microdilution method using EUSVEC3 plates. Overall, 4,87% (18/369) of the individuals were *Salmonella* carriers, with *L. fuscus* showing the highest prevalence (7,4%) followed by *L. michahellis* (3,4%). A high diversity of serovars was identified, with each gull species carrying different serovars: Bovismorbificans, Derby, Isangi, London, and Poona, in *L. fuscus*; Virchow, Brandenburg, Typhimurium monophasic, and Stanley in *L. michahellis*. Overall, 22,22% (4 out of 18) of *Salmonella* isolates were resistant to at least one antimicrobial agent and belonged to serovars Virchow and monophasic *S. Typhimurium* for *L. michahellis*, and ser. Bovismorbificans for the single resistant isolate from *L. fuscus*. The single multidrug resistant strain was recovered from *L. michahellis* and belonged to the serovar monophasic *S. Typhimurium*, with the AMR profile ampicillin-tetracycline-sulfamethoxazole. We have also commonly found this AMR profile in isolates from monophasic *S. Typhimurium* carried by *L. michahellis* from a densely populated colony in northeastern Spain. Besides, of the two gull species sampled, *L. michahellis* carried three out of the four AMR strains found. Despite the relatively low prevalence of *Salmonella* in both gull species, it is noteworthy the prevalence of AMR strains. These results highlight gulls as potential spreaders of AMR strains in the environment that may pose a risk for public health, especially in human populated areas, as also shown in other gull colonies in southern Europe [1]. It also supports the potential role of gulls as sentinels for AMR in the environment.

## References

[1] N. Antilles, I. García-Bocanegra, A. Alba-Casals, S. López-Soria, N. Pérez-Méndez, et al. (2021). *Sci Total Environ.* 763:143018.

[2] R. Ramos, M. Cerdà-Cuéllar, F. Ramírez, L. Jover, and X. Ruiz, (2010). *Applied Environ Microb.* 76:3052–3056.

<footnote n="1">¹Unitat mixta d'Investigació IRTA-UAB en Sanitat Animal. Centre de Recerca en Sanitat Animal (CReSA). Campus de la Universitat Autònoma de Barcelona (UAB), Bellaterra, 08193, Catalonia. Spain.</footnote>

<footnote n="2">²IRTA. Programa de Sanitat Animal. Centre de Recerca en Sanitat Animal (CReSA). Campus de la Universitat Autònoma de Barcelona (UAB), Bellaterra, 08193, Catalonia. Spain.</footnote>

<footnote n="3">³Instituto Español de Oceanografía, Centro Oceanográfico de Málaga – CSIC, Puerto pesquero sn, Fuengirola, 29640, Spain</footnote>

<footnote n="4">⁴Institut de Recerca de la Biodiversitat (IRBio) and Departament de Biologia Evolutiva, Ecologia i Ciències Ambientals, Universitat de Barcelona, Av Diagonal 643, Barcelona 08028, Spain.</footnote>
# Ambulatory decolonization of a cat carrying Panton-Valentine leukocidin (PVL)-producing *S. aureus*

Astrid Bethe<fnref n="1" />,<fnref n="2" />, Anne-Kathrin Schink<fnref n="1" />,<fnref n="3" />, Julian Brombach<fnref n="1" />,<fnref n="2" />, Susanne Reinhardt<fnref n="4" />,
Ernst Molitor<fnref n="5" />, Svenja Müller<fnref n="5" />, Julian Balks<fnref n="5" />, Robin Köck<fnref n="6" />, Stefan Schwarz<fnref n="1" />,<fnref n="2" />, Birgit Walther⁷,
Antina Lübke-Becker<fnref n="1" />,<fnref n="2" />

**Introduction:** Panton-Valentine leukocidin (PVL)-producing *Staphylococcus aureus* (PVL-SA) is a frequent cause of skin abscesses in humans. Besides antibiotics and surgical treatment, topical decolonization is a preventive measure for reducing the risk of PVL-SA recurrence and transmission. Here, we report on an affected family of four that was treated but continued to suffer from repeated PVL-SA positive skin infections even after three courses of appropriate decolonization and whose two cats were identified to be colonized by *S. aureus* (SA). Although dogs and cats can be potential carriers of PVL-SA and hence a source of reinfection for humans, validated protocols for decolonization of animals are not available. Thus, a protocol for outpatient decolonization using systemic antibiotic treatment of cats was developed.

**Methods:** For bacteriological examination, samples from both cats were obtained from the nose, oral cavity, rectum, inguinal and perianal region. SA isolates were tested for the presence of *lukF-lukS* by PCR and subjected to susceptibility testing (AST) according to CLSI. Comparative analysis of human and feline isolates was performed by whole genome sequencing. Results of susceptibility testing were used to develop a decolonization protocol based on systemic therapy with amoxicillin-clavulanic acid for 10 d and 20 d, respectively.

**Results:** Methicillin-susceptible SA was isolated from the oral cavity and nose of both cats. While one cat was a carrier of PVL-SA, a PVL-negative SA strain was isolated from the second cat. Comparative whole genome analysis revealed close clonal relationships of both the PVL-SA assigned to sequence type (ST 8) and a further SA (ST45) isolated from human and feline samples. In the course of oral decolonization therapy, a significant reduction of SA was accomplished. Although the samples of the SA-carrying cat were negative after 10 days, the PVL-SA-positive cat required a second course of antibiotics (amoxicillin for 18 days). Control examinations of the cats after 3 and 7 weeks were negative for SA.

**Discussion:** In the case presented, successful decolonization of cats colonized with PVL-positive or -negative *S. aureus* was achieved by a combination of systemic antibiotic therapy and hygiene measures. The close relationship of human and feline isolates suggests transmission between humans and animals in the household and underscores the importance of potentially colonized pets for the success of decolonization measures.

<footnote n="1">¹Institute for Microbiology and Epizootics, Freie Universität Berlin, Germany,</footnote>

<footnote n="2">²Veterinary Centre for Resistance Research (TZR), Freie Universität Berlin, Germany,</footnote>

<footnote n="3">³LDG Laboratory Diagnostics Germany GmbH, Germany,</footnote>

<footnote n="4">⁴Kleintierpraxis am Kenntemich-Platz, Troisdorf, Germany,</footnote>

<footnote n="5">⁵Institute for Medical Microbiology, Immunology, and Parasitology, University Hospital Bonn, Germany,</footnote>

<footnote n="6">⁶Hygiene and Environmental Medicine, Universitätsmedizin Essen, Germany, ⁷Microbiological Risks, Division II, section 1.4, Umweltbundesamt Berlin, Germany</footnote>
# Starved of ACTION: A Critical Look at the Antimicrobial Resistance Action Plans of African Countries

Godwin Pius OHEMU

Microbiology Technology, School of Science Laboratory Technology, University of Port Harcourt, Choba, East-West Road, Port Harcourt 500102, Nigeria

The urgent need for Africa, as a continent, to start galvanizing resources and strengthening its capacity to win the fight against the looming threat of antimicrobial resistance (AMR) was once again brought to the limelight by the recent study on the "[Global Burden of Bacterial Antimicrobial Resistance](https://wwwPMC.org/abstract/10.1016/S0140-6736(22)00091-5)" (The Lancet 2022, 399, 629). According to the study, western sub-Saharan Africa is a super-region for AMR, with overall mortality rates due to and associated with AMR standing at 27.3 and 114.8 per 100 000 people, respectively. Putting this in perspective, if the leaders of this region do not take effective action, Africa will indeed be one of the two worst-affected regions, along with Asia, with the possibility of over 4.1 million people dying annually from AMR by 2050. The development of a National Action Plan, as directed by the World Health Organization, provides an important framework for addressing the complex and multi-faceted nature of the rise and spread of drug-resistant bacteria. This paper reviews the AMR National Action Plans of African countries and calls on the leaders of this region to move from paper to action and to look beyond just the reduction of antibiotic consumption and, as a more comprehensive response to the threat of AMR, focus also on expanding access to safe drinking water, bettering sanitary conditions, maintaining effective leadership at all levels of governance, increasing government spending on healthcare services, and strengthening regulatory oversight.

## Reference

[1] The Lancet Antimicrobial resistance: time to repurpose the Global Fund. *The Lancet* **2022**, *399* (10322), 335, DOI: 10.1016/S0140-6736(22)00091-5 [Crossref], [PubMed], [CAS], [Google Scholar](https://GoogleScholar.com)
# Multidirectional dynamic model for the spread of Extended-Spectrum-β-lactamase-Producing *E. coli* in the Netherlands

Eduardo F. COSTA<fnref n="1" />, Thomas HAGENAARS<fnref n="1" /> Anita DAME-KOREVAAR<fnref n="1" />, Michael BROUWER<fnref n="2" />, Clazien J. de VOS<fnref n="1" />

This work aimed to design a multidirectional dynamic risk model with which the contribution of different sources to the extended-spectrum β-lactamase *Escherichia coli* (ESBL-EC) prevalence in humans can be assessed. A discrete-time model was built to describe within and between population spread of ESBL-EC in humans (open community), farmers, broiler flocks and veal herds. The slaughterhouse and consumer phase (food preparation) were included in the model to account for ESBL-EC transmission via food consumption. Recreational surface water was added as a source of ESBL-EC for humans when swimming in surface water. The model was parameterized for the Netherlands. Data availability was limited and a sensitivity analysis was conducted to explore the effect of parameter uncertainty on model results. The average ESBL-EC prevalence in the open community, broiler farmers and veal farmers after simulating transmission for 200 weeks was 2.1%, 20.1% and 47.3%, respectively. The most relevant sources for ESBL-EC colonization of the open community were the open community itself, contact of the open community with veal farmers, and consumption of vegetables cross-contaminated by chicken meat with 59.4%, 18.5%, and 12.5%, respectively (Fig. 1). The uncertain parameters that most affected model results were the colonization and decolonization rates for humans.

<figure><img src="image_65.png" /><figcaption>**Fig. 1.** Relative source attribution to the different populations included in the multidirectional model for ESBL-EC spread in the Netherlands. NA: source not considered for this population.</figcaption></figure>

Results of the model contribute to a better understanding of the relative importance of sources contributing to ESBL-EC prevalence in humans. The model, however, also accounts for humans' contribution to the ESBL-EC prevalence in these sources, resulting in a One Health approach for risk assessment of antimicrobial resistance transmission. The model results can inform public health policymakers to mitigate antimicrobial resistance risk.

<footnote n="1">¹Department of Epidemiology, Bioinformatics and Animal Models, Wageningen Bioveterinary Research, Houtribweg 39, 8221 RA Lelystad, the Netherlands</footnote>

<footnote n="2">²Department of Bacteriology, Host Pathogen Interaction & Diagnostics Development, Wageningen Bioveterinary Research, Houtribweg 39, 8221 RA Lelystad, the Netherlands.</footnote>
# Focus on data and modelling practice – a systematic review of AMR transmission model.

Maja L. BRINCH¹, Andrea PALLADINO², Jeroen GEURTSEN³, Thierry VAN EFFELTERRE³, and Tine HALD¹

¹Foodborne Pathogens and Epidemiology, DTU National Food Institute, Kgs Lyngby, Denmark

²GSK Biologicals SA, Siena, Italy

³Janssen Vaccines & Prevention, Leiden, The Netherlands

Mathematical transmission models are a valuable tool to guide intervention strategies in public health. Literature reports a number of systematic reviews on antimicrobial resistance (AMR) transmission models with different aims. Here we systematically search, and review publications modelling the transmission of antimicrobial-resistant bacterial pathogens in the human population and evaluate models by the TRACE framework and thereby identify gaps in the modelling of AMR.

A total of 168 studies were identified and included in the review. The most targeted WHO regions were the European region and the region of the Americas. *Mycobacterium tuberculosis* (n = 40) and *Staphylococcus aureus* (n = 38) were the most modelled pathogens, with a focus on multi-drug and methicillin resistance, respectively. A large fraction of the studies modelled the effects of interventions, hereof 1/3 with more than one intervention. Half of the studies modelled symptomatic infections, here most of them specified a specific infection site. Most of the included studies were population-based compartment models (n = 110). Models describing validation and sensitivity analysis (n = 39) were evaluated by applying the TRACE framework. The evaluation showed a general lack of validation of models both internally and with external data. Our review highlights the importance of describing good modelling practices and the need for more data to compare with the model outputs.
<figure><img src="image_66.png" /></figure>

# PROMISE, a One Health meta-network to fight antibiotic resistance in France

Yohann Lacotte<fnref n="1" />, Elodie Pfender<fnref n="2" />, Rémi Manczak<fnref n="2" />, Olivier Barraud<fnref n="1" />, Christophe Dagot<fnref n="1" />, Bruno François<fnref n="1" />,<fnref n="2" />, Marie-Cécile Ploy<fnref n="1" />

## Introduction and objectives:

Antimicrobial resistance (AMR) is a major health issue affecting humans, animals and ecosystems. To fight effectively against AMR, we need to adopt a One Health approach taking into account the interconnection between these 3 sectors. However, the operationalization of this concept often remains difficult due to the differences between these 3 worlds.

Launched in 2021 and supported by the French priority research program on AMR, the PROMISE project (https://amr-promise.fr/fr/) brings together the main actors involved in the fight against AMR in France (25 professional networks and more than 40 research units, from the 3 sectors) to build a large One Health community on AMR. PROMISE aims to build interactions between actors usually working in silo and allows its members to coordinate their actions.

## Methods:

PROMISE's action is based on 7 transdisciplinary pillars:

* Knowledge and skills sharing

* Data sharing

* AMR surveillance

* Clinical research

* Communication and awareness

* Training

* Structuring of emerging networks in areas lacking coordination

## Results, conclusion:

The main results of PROMISE include:

* A cross-sectoral analysis of the correlations between consumption and resistance in France;

* The creation of a common One Health data warehouse gathering surveillance data from the 3 sectors and paving the way for a more integrated AMR surveillance.

* The launch of large-scale clinical studies involving the 4 main clinical research networks of intensive care units and infectious diseases in France;

* The organization of events allowing the community to come together;

* The launch of a diploma course allowing professionals from the 3 sectors to learn about AMR and One Health

* The coordination of a new network dedicated to the operationalization of a AMR monitoring system in the environment

* The structuration of a network dedicated to preclinical research in antibiotic therapy

To conclude, PROMISE is a unique opportunity to structure the AMR community and make France a best practice region in Europe in terms of One Health coordination.

<footnote n="1">¹Univ. Limoges, Inserm, CHU Limoges, RESINFIT, U 1092, F-87000 Limoges, France.</footnote>

<footnote n="2">²CHU Limoges, CIC1435, F-87000 Limoges, France</footnote>
# Coalition of Non-Profit Organizations Pushing for Policy Change in the United States' Industrial Agriculture Sector

Madeleine Kleven

The Keep Antibiotics Working Coalition
Food Animal Concerns Trust/Chicago, IL, USA

The Keep Antibiotics Working (KAW) Coalition fights against the spread of antibiotic-resistant superbugs. Formed in 2001, KAW is a coalition of nineteen advocacy groups that joined to ensure that untreatable superbugs resulting from the overuse of antibiotics on farms do not reverse the medical advances of the past century. KAW urges that it is more critical than ever for the United States Food and Drug Administration (FDA) and the United States Department of Health and Human Services (HHS) to confront the slow-moving resistance pandemic, which often spreads via animals and is exacerbated by livestock husbandry practices. We need information about how and why antibiotics are used across all settings, in order to fully understand and respond to ongoing antibiotic overuse – one of the largest, if not the largest driver of the spread of resistance. Curbing medically important antibiotic use in food-producing animals is especially important; around two-thirds of the nation’s medically important drugs are sold for use on animals, not people. [1] The FDA must begin to robustly monitor antibiotic use, but also prioritize actions to effectively curb the overuse of these precious medicines. KAW organizations advocate for government action to control the overuse of on-farm antibiotics. Specifically, we recommend that the FDA and HHS commit to the following actions to protect public health.

1. Establish a national stewardship goal to curb medically important antibiotic sales for use in food-producing animals by at least 50% by the end of 2025, relative to 2010 levels.

2. End administration of antibiotics to entire herds where there are not sick animals. The U.S. finished phasing out antibiotics for growth promotion in 2017, but use of the same drugs in healthy animals for disease prevention continues to be legal. In order to promote stewardship preventive use should be disallowed, except in the case of surgery. This will slow the emergence of antibiotic resistance and encourage higher welfare standards in farming operations.

3. Develop comprehensive, national systems to monitor and report pertaining to farm-level antibiotic use and resistance, using a One Health approach. The continued lack of national systems to track antibiotic use at the farm level leaves a critical gap in antibiotic stewardship efforts and national pandemic preparedness.

4. KAW expects farms to prevent disease by providing appropriate diets, delaying weaning, improving hygiene, reducing crowding, and using vaccines appropriately-so that antibiotic use on farm is rare not routine.

## References

[1] David Wallinga, Eili Klein, and Alisa Hamilton, "U.S. Livestock Antibiotic Use is Rising, Medical Use Falls," November 18, 2021.
# Contribution to the control of antimicrobial resistance in human and animal health in Togo: a One Health approach

Esso-tchella Madéra BODOMBOSSOU, Alexis DELABOUGLISE, Andrea APOLLONI, Éric CARDINALE

UMR ASTRE, CIRAD/Montpellier, France

Antibiotic resistance among bacteria in West Africa is a major concern. In Togo, no studies have precisely characterized isolated multidrug-resistant bacteria or compared their genomes to identify transmission pathways. It is within this context that our study is conducted.

During this study, we assess the prevalence of carriage of extended-spectrum β-lactamase-producing Enterobacteriaceae (ESBL) and carbapenemase-producing Enterobacteriaceae (CPE) in farmers and their animals, as well as the possibility of inter-species transmission of strains. To achieve this, samples are collected from farmers and their animals to isolate and compare phenotypically and genotypically the resistant bacterial strains. Additionally, an epidemiological and socio-economic survey conducted in parallel allows us to potentially highlight risk factors associated with inter-species transmission and evaluate the acceptability of a rational antibiotic use plan in livestock farming.

The main objective of our study is to estimate the prevalence of ESBL and CPE carriage in farmers and their animals living in the maritime region of Togo. The secondary objectives are to identify risk factors associated with ESBL and CPE carriage in animals, characterize the species producing ESBL and CPE, as well as the enzymes responsible for resistance, and assess the acceptability of a plan to reduce antibiotic use in livestock farming.

The methods of our study involve administering a questionnaire to farmers to list the risk factors for the emergence of ESBL/CPE in livestock farming and assess the farmer's level of investment in antimicrobial use and their willingness to consider alternative methods for controlling animal diseases through a semi-structured interview. All farmers who refuse to participate in the study are excluded. Fecal samples are collected from 160 animal productions, as well as 80 stool samples from farmers, either by rectal swabbing or using a stool potty. The detection of ESBL and CPE is performed on MacConkey agar supplemented with cefotaxime and MacConkey agar supplemented with ertapenem, respectively. Bacterial colonies are identified using mini-galleries or a complete gallery (API 20E). Antibiotic susceptibility testing is also conducted. The identification of resistance mechanisms is performed using either phenotypic methods (ESBL) or genotypic methods (Genexpert, Cepheid for CPE). Genomic analyses are also carried out through whole-genome sequencing (Miniseq, Illumina). Regarding the results, the survey is currently still ongoing. However, ESBL and CPE have been isolated in 91.9% and 8.1% of the first 37 investigated farms, respectively. Among the multidrug-resistant bacteria (MDR) present in the farms, a significant proportion of *E. coli* (54.1%) and *Raoutella ornitholytica* (13.5%) have been observed.
With the generous grants from

INRAO

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Région

Centre-Val de Loire

UT university de TOURS

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SFM

Société Française de Microbiologie

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Federation of European Microbiological Societies

Frontiers in Microbiology

interscience pour la microbiologie

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re sistomap

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Organised by
UMR 1282 Infectiologie et Santé Publique
Institut National de Recherche pour l'Agriculture, l'Alimentation et l'environnement

INRA
science for people, life & earth