# Investigation of urban birds as source of β-lactamase-producing Gram-negative bacteria in Marseille city, France

Edgarthe Priscilla Ngaiganam, Isabelle Pagnier, Wafaa Chaalal, Thongpan Leangapichart, Selma Chabou, Jean-Marc Rolain, Seydina Mouhamadou Diene

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Edgarthe Priscilla Ngaiganam, Isabelle Pagnier, Wafaa Chaalal, Thongpan Leangapichart, Selma Chabou, et al.. Investigation of urban birds as source of β-lactamase-producing Gram-negative bacteria in Marseille city, France. Acta Veterinaria Scandinavica Supplementum, 2019, 61 (1), 10.1186/s13028-019-0486-9. hal-02458101

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# Investigation of urban birds as source of β-lactamase-producing Gram-negative bacteria in Marseille city, France

Edgarthe Priscilla Ngaiganam<fnref n="1" />, Isabelle Pagnier<fnref n="1" />,², Wafaa Chaalal<fnref n="1" />, Thongpan Leangapichart<fnref n="1" />, Selma Chabou<fnref n="1" />, Jean-Marc Rolain<fnref n="1" />,² and Seydina Mouhamadou Diene<fnref n="1" />,²*

## Abstract

**Background:** We investigate here the presence of multidrug-resistant bacteria isolated from stool samples of yellow-legged gulls and chickens (n = 136) in urban parks and beaches of Marseille, France. Bacterial isolation was performed on selective media, including MacConkey agar with ceftriaxone and LBJMR medium. Antibiotic resistance genes, including extended-spectrum β-lactamases (ESBL) (i.e. *bla*<sub>CTX-M</sub>, *bla*<sub>TEM</sub> and *bla*<sub>SHV</sub>), carbapenemases (*bla*<sub>KPC</sub>, *bla*<sub>VIM</sub>, *bla*<sub>NDM</sub>, *bla*<sub>OXA-23</sub>, *bla*<sub>OXA-24</sub>, *bla*<sub>OXA-48</sub> and *bla*<sub>OXA-58</sub>) and colistin resistance genes (*mcr*-1 to *mcr*-5) were screened by real-time PCR and standard PCR and sequenced when found.

**Results:** Of the 136 stools samples collected, seven ESBL-producing Gram-negative bacteria (BGN) and 12 colistin-resistant *Enterobacteriaceae* were isolated. Among them, five ESBL-producing *Escherichia coli* and eight colistin-resistant *Hafnia alvei* strains were identified. Four *bla*<sub>TEM-1</sub> genes were detected in yellow-legged gulls and chickens. Three CTX-M-15 genes were detected in yellow-legged gulls and pigeons, and one CTX-M-1 in a yellow-legged gull. No *mcr*-1 to *mcr*-5 gene were detected in colistin-resistant isolates. Genotyping of *E. coli* strains revealed four different sequence types already described in humans and animals and one new sequence type.

**Conclusions:** Urban birds, which are believed to have no contact with antibiotics appear as potential source of ESBL genes. Our findings highlight the important role of urban birds in the proliferation of multidrug-resistant bacteria and also the possible zoonotic transmission of such bacteria from wild birds to humans.

**Keywords:** Chickens, Extended-spectrum beta-lactamases (ESBL), Marseille, Pigeons, Urban birds, Yellow-legged gulls

## Background

Extended-spectrum beta-lactamases (ESBLs) are mainly plasmid-encoded enzymes that confer resistance to beta-lactams, including broad spectrum cephalosporins. They can be transmitted between bacteria by horizontal gene transfer (HGT) via mobile genetic elements such as recombinant plasmids, transposons or integrons [1]. These ESBL enzymes mainly include class A β-lactamases, such as TEM, SHV and CTX-M type, that confer resistance to penicillin and cephalosporin classes. However, CTX-M β-lactamase enzymes are

the most emerged and reported worldwide [1]. ESBL-producing *Enterobacteriaceae* cause infections in both humans and animals through contaminations of water and food [2]. Over the past decade, a high prevalence of antibiotic resistance genes has been reported and has stimulated the search for multidrug-resistant bacteria in poultry, pigeons [3, 4] and birds in different countries [5]. In recent years, several reports indicated the presence of ESBL-producing bacteria among different species of urban and wild birds with no apparent exposure to antimicrobial drugs [6]. As reported by Bonnedahl et al. [7], 9.4% of yellow-legged gulls (*Larus michahellis*) carried CTX-M type ESBL-producing *E. coli* in the South of France. Recently, Stedt et al. [8] have reported high and alarming levels of CTX-M genes in species of gulls

<footnote>*Correspondence: seydina.diene@univ-amu.fr</footnote>

<footnote n="1">¹IRD, APHM, MEPHI, IHU-Mediterranee Infection, Aix-Marseille Univ, 19-21</footnote>

<footnote>bd Jean Moulin, 13005 Marseille, France</footnote>

<footnote>Full list of author information is available at the end of the article</footnote>
in Europe and indicated that the ability of these birds to reach several countries could contribute to the spread of ESBLs. Therefore, pigeons and gulls, considered as migratory birds, have been pointed out as potential reservoirs and vectors for multidrug-resistant bacteria [8–10], especially gulls that are free-living aquatic birds. More recently, Wang et al. [11] have reported the role of wild birds in the global transmission of ESBL, AmpC β-lactamase, carbapenemase, and colistin resistance genes in *Enterobacteriaceae*. Because of their frequent movement in landfills and wastewater and the ease with which they collect food from various places, they can contribute to fecal contamination of natural water reservoirs or the food they come in contact with [5, 12]. Face to this phenomenon, we aim here to investigate and to characterize β-lactamase-producing bacteria and

colistin-resistant bacteria isolated from urban birds in the city of Marseille, France.

## Methods

### Sample collection

Between July and September 2016, six parks located respectively in four different districts of Marseille city, and one beach located in the 16th district of Marseille, were investigated (Fig. 1 and Additional file 1). A total of 136 stool samples from 71 pigeons (*Columba livia f. urbana*), 28 chickens (*Gallus gallus domesticus*), and 37 yellow-legged gulls (*Larus michahellis*), were collected (Fig. 1 and Additional file 1). About 10 g of feces per sample was collected from the ground using sterile forceps and transferred into sterile 1.5 mL Eppendorf tubes. The samples were then immediately placed

<figure><img src="image_3.png" /></figure>
on ice during transport. Microbiological analyses were
then carried the day after sampling.

DNA extraction and ESBL detection

Genomic DNA from 136 feces samples was prepared
from an overnight incubation at 56 °C and extracted
with the EZ1 DNeasy Blood Tissue kit (Qiagen GmbH,
Hilden, Germany) using the protocol of the manu-
facturer. The presence of genes encoding for ESBLs
(CTX-M, TEM and SHV, PER, GES, and VEB), car-
bapenemases (KPC, VM, NDM, OXA-23, OXA-24,
OXA-48 and OXA-58) and mcr-1 to mcr-5 genes
were investigated by real-time polymerase chain reac-
tion (RT-PCR) with the specific primers as previously
described [13–16]. Moreover, we designed here prim-
ers and probe for detection of All blaTEM gene vari-
ants (ALLTEM_RT_F: 5'-TTCTGCTATGTGGTGCGG
TA-3'; ALLTEM_RT_R: 3'-GTCCTCCGATCGTTG
TCAGA-5'; ALLTEM_RT_Probe: 5'-AACTCGGTC
GCCGCATACACTATTCTCAGA-3').

Isolation and identification of bacterial strains

To isolate β-lactamase-producing bacteria and colistin
resistant bacteria, 1 g of feces samples was mixed with
1 mL sterile water and 100 µL were enriched in Tryp-
tic Soy Broth (BBL™ Trypticase™ Soy Broth, Becton,
Dickinson and Company Sparks, MD 21152, USA) and
incubated overnight at 37 °C for preliminary analysis. RT-
PCR targeting β-lactamase genes were used to screen all
samples for positive ESBL genes. When a positive sam-
ple was suspected of isolates producing ESBL genes,
bacteria were cultured on MacConkey agar medium
with added ceftriaxone (2 µg/mL). On the other hand,
in order to look for colistin resistant bacteria, all sam-
ple were directly cultured on the selective Lucie Bar-
det-Jean-Marc Rolain (LBJMR) medium [17] containing
4 µg/mL of colistin and 50 µg/mL of vancomycin. All cul-
tures were then incubated at 37 °C for 24 h. From one to
eight colonies of different morphologies were collected
in each medium and reisolated on Trypticase Soy Agar
(TSA, BioMerieux SA, France). Each of these colonies
was subcultured 3 additional times to obtain a monocul-
ture. Isolates were then identified by matrix assisted laser
desorption/ionization, time of flight mass spectrometry
(MALDI-TOF MS) and MALDI-Biotyper 3.0 software
(Bruker Daltonics, Bremen, Germany) as described pre-
viously [18]. Bacteria are efficiently identified at the genus
and species level when score values are ranging from 2.3
to 3.0 and at the secure genus identification and probable
species identification when score values are ranged from
2.0 to 2.3 [18].

Antimicrobial susceptibility testing

Antibiotic susceptibility testing (AST) was performed using the agar disk diffusion method. Bacterial strains (n=19) isolated from birds were suspended in a saline solution to a density of 0.5 McFarland. The surface of Mueller–Hinton agar plates (Merck, Darmstadt, Germany) was inoculated and the antibiotic disks were placed. After aerobic incubation at 37 °C for 18–22 h, bacterial resistance was determined by measuring the inhibition zone according to the guidelines of CASSFM/EUCAST 2019 (v.1.0) (https://www.sfm-microbiologie.org/wp-content/uploads/2019/02/CASFM2019_V1.0.pdf). The following antibiotics were tested (µg/disk): amoxicillin (20), amoxicillin–clavulanic acid (20/10), cefepime (30), piperacillin–tazobactam (30/6), cefalotin (30), ceftriaxone (30), ertapenem (10), imipenem (10), fosfomycin (200), nitrofuran (100), trimethoprim–sulfamethoxazole (1.25/23.75), amikacin (30), ciprofloxacin (5), colistin (50), and gentamicin (10). For bacteria isolated on LBJMR medium, colistin MICs were determined by microdilution method using Mueller–Hinton (MH) liquid medium.

Sequence analysis and MLST

Aminoglycoside resistance genes (i.e. *aad*, *ant*, *aph*,
*aac(6')-Ib*, *aac(3')*, *armA*, and *aac(6')-Ib-cr*) were
investigated in *Escherichia coli* isolate (B32 strain)
resistant to gentamicin and doxycycline. On overall, by
qPCR and standard PCR we investigated genes encod-
ing for extend-spectrum-β-lactamases (*bla*<sub>SHV</sub>, *bla*<sub>VEB</sub>,
*bla*<sub>GES</sub>, *bla*<sub>PER</sub>), carbapenemases (VIM, NDM, OXA-23,
OXA-24, OXA-48, OXA-58), and mobile colistin resist-
ance genes (*mcr-1* to *mcr-5*) using primers and probes
previously reported [19].

All positive PCR products for genes encoding for
ESBL that conferred resistance to 3rd generation
cephalosporins were purified using the QIAquick PCR
purification kit in concordance with the manufactur-
er's instructions (Qiagen, Hilden, Germany). Sequenc-
ing was performed using the BigDye Terminator Cycle
Sequencing kit and an automated fluorescent DNA
sequencer ABI 3730xl (Applied Biosystems, Foster
City, CA, USA) in accordance with the manufactur-
er's instructions. The nucleotide sequences obtained
were compared with those from the NCBI database by
BlastN analysis. Multilocus sequence typing (MLST)
was performed for five ESBL-producing *E. coli* isolates
using genes from both the Warwick (*adk*, *fumC*, *gyrB*,
*icd*, *mdh*, *purA*, and *recA*) (http://mlst.warwick.ac.uk/
mlst/dbs/Ecoli) and Pasteur Institute (*icd*, *pabB*, *polB*,
*putP*, *trpA*, and *trpB*) (http://www.pasteur.fr/reche
rche/genopole/PF8/mlst/) schemes. The ST not found
in the database was submitted to the Pasteur MLST database to define the new profile.

## Results

From the 136 stool samples from 71 pigeons (*Columba livia* f. urbana), 28 chickens (*Gallus gallus domesticus*), and 37 yellow-legged gulls (*Larus michahellis*), 15 samples were positive by the RT-PCR targeting ESBL-encoding genes. The culture of these samples on the MacConkey with ceftriaxone medium reveals seven isolates from six samples (Table 1). On the selective LBJMR medium, the culture results to 12 isolates from 12 samples (Table 1). From the 19 isolated strains, bacterial identification by MALDI-TOF revealed, with a score ranging between 2.15 and 2.95, the presence of *E. coli* (n=5), *Cronobacter sakazakii* (n=1), *Pseudomonas aeruginosa* (n=1), *Hafnia alvei* (n=8), *Proteus hauseri* (n=1), *Panteoa ananatis* (n=1), *Providencia alcalifaciens* (n=1) and *Serratia marcescens* (n=1).

Only bacterial strains isolated on the MacConkey agar exhibited reduced susceptibility to 3rd generation cephalosporin (ceftriaxone) and only *E. coli* strains were resistant to cefepime (4th generation cephalosporin). Resistance also to trimethoprim-sulfamethoxazole, ciprofloxacin, doxycycline, and gentamicin was observed in some of these isolates (Table 1). However, all these strains from this MacConkey agar remained susceptible to carbapenems and colistin. For 3rd generation cephalosporin-resistant isolates, the standard PCR performed and sequencing revealed the presence of ESBL genes such as *bla*<sub>CTX-M</sub> and/or *bla*<sub>TEM</sub> with amino acid similarity ranging from 99.61 to 100% (Additional file 1). Four genes encoding two CTX-M variants were identified (Table 1). The *bla*<sub>CTX-M-15</sub> gene was found in three *E. coli* isolates from two yellow-legged gulls and one pigeon. Only one *E. coli* isolate from a yellow-legged gull harboured simultaneously *bla*<sub>CTX-M-1</sub> and *bla*<sub>TEM-1</sub>. Moreover, four *bla*<sub>TEM-1</sub> β-lactamase genes were detected in two

<table><caption>Table 1 General features of all bacterial strains isolated from urban birds from the different investigated sites of Marseille city</caption><thead><tr><th>Strain name</th><th>Isolates</th><th>Sources</th><th>Sites</th><th>Antibiotic resistance phenotype</th><th>MIC<sub>CT</sub> (µg/ml)</th><th>bla<sub>CTX-M</sub> gene<sup>a</sup></th><th>bla<sub>TEM</sub> gene<sup>a</sup></th><th>MLST</th></tr></thead><tbody><tr><td colspan="9"><strong>Bacterial strains isolated from the MacConkey agar</strong></td></tr><tr><td>B11</td><td>Escherichia coli</td><td>Yellow-legged gull</td><td>Site 1</td><td>AX, AMC, CRO, FEP, STX, CIP</td><td>0.25</td><td>CTX-M-15</td><td>–</td><td>ST10</td></tr><tr><td>B32</td><td>Escherichia coli</td><td>Chicken</td><td>Site 2</td><td>AX, AMC, CRO, FEP, STX, CN</td><td>0.25</td><td>–</td><td>TEM-1</td><td>ST1652</td></tr><tr><td>B60</td><td>Escherichia coli</td><td>Yellow-legged gull</td><td>Site 3</td><td>AX, AMC, CRO, FEP, STX</td><td>0.25</td><td>CTX-M-15</td><td>–</td><td>ST38</td></tr><tr><td>A61</td><td>Escherichia coli</td><td>Pigeon</td><td>Site 2</td><td>AX, KF, CRO, FEP</td><td>0.25</td><td>CTX-M-15</td><td>–</td><td>ST34</td></tr><tr><td>B62</td><td>Escherichia coli</td><td>Yellow-legged gull</td><td>Site 3</td><td>AX, AMC, CRO, FEP</td><td>0.25</td><td>CTX-M-1</td><td>TEM-1</td><td>ST857</td></tr><tr><td>B62</td><td>Cronobacter sakazakii</td><td>Yellow-legged gull</td><td>Site 3</td><td>AX, AMC, CRO, FF</td><td>0.25</td><td>–</td><td>TEM-1</td><td>–</td></tr><tr><td>B34</td><td>Pseudomonas aeruginosa</td><td>Chicken</td><td>Site 2</td><td>AX, KF, CRO, F, STX</td><td>0.25</td><td>–</td><td>TEM-1</td><td>–</td></tr><tr><td colspan="9"><strong>Bacterial strains isolated from the LBJMR agar</strong></td></tr><tr><td>B42</td><td>Hafnia alvei</td><td>Chicken</td><td>Site 2</td><td>AX, AMC, KF, CT</td><td>8</td><td>–</td><td>–</td><td>–</td></tr><tr><td>B02</td><td>Hafnia alvei</td><td>Yellow-legged gull</td><td>Site 1</td><td>AX, AMC, KF, CT</td><td>4</td><td>–</td><td>–</td><td>–</td></tr><tr><td>B04</td><td>Hafnia alvei</td><td>Yellow-legged gull</td><td>Site 1</td><td>KF, CT</td><td>4</td><td>–</td><td>–</td><td>–</td></tr><tr><td>B11</td><td>Hafnia alvei</td><td>Yellow-legged gull</td><td>Site 1</td><td>AX, AMC, KF, CT</td><td>4</td><td>–</td><td>–</td><td>–</td></tr><tr><td>B21</td><td>Hafnia alvei</td><td>Yellow-legged gull</td><td>Site 4</td><td>KF, CT</td><td>4</td><td>–</td><td>–</td><td>–</td></tr><tr><td>B47</td><td>Hafnia alvei</td><td>Chicken</td><td>Site 2</td><td>AX, AMC, KF, CT</td><td>4</td><td>–</td><td>–</td><td>–</td></tr><tr><td>B59</td><td>Hafnia alvei</td><td>Yellow-legged gull</td><td>Site 3</td><td>AX, AMC, KF, FF, F, CT</td><td>4</td><td>–</td><td>–</td><td>–</td></tr><tr><td>A63</td><td>Hafnia alvei</td><td>Pigeon</td><td>Site 2</td><td>AX, AMC, KF, FF, F, CT</td><td>4</td><td>–</td><td>–</td><td>–</td></tr><tr><td>A61</td><td>Panteoa ananatis</td><td>Pigeon</td><td>Site 2</td><td>AX, AMC, KF, FF, F, CT</td><td>> 256</td><td>–</td><td>–</td><td>–</td></tr><tr><td>B05</td><td>Proteus hauseri</td><td>Yellow-legged gull</td><td>Site 1</td><td>F, CT</td><td>> 256</td><td>–</td><td>–</td><td>–</td></tr><tr><td>B22</td><td>Serratia marcescens</td><td>Yellow-legged gull</td><td>Site 4</td><td>F, CT</td><td>> 256</td><td>–</td><td>–</td><td>–</td></tr><tr><td>B36</td><td>Providencia alcalifaciens</td><td>Chicken</td><td>Site 2</td><td>F, CT</td><td>> 256</td><td>–</td><td>–</td><td>–</td></tr></tbody></table>

Site 1: Palais Longchamp; Site 2: Parc 26e Centenaire; Site 3: Plage Estaque; Site 4: Parc Borely

AX amoxicillin, AMC amoxicillin-clavulanic acid, KF cefalotin, CRO ceftriaxone, FEP cefepime, ERT ertapenem, IMP imipenem, FF fosfomycin, F nitrofurantoin, SXT trimethoprim-sulfamethoxazole, CIP ciprofloxacin, CT colistin, CN gentamycin, ST sequence type

<footnote><sup>a</sup> Obtained β-lactamase sequences were compared with reference protein sequences of bets hit sequences which were *bla*<sub>CTX-M-15</sub> (JQ686199); *bla*<sub>CTX-M-1</sub> (X92506); and *bla*<sub>TEM-1</sub> (JQ735917) (cf. Additional file 2)</footnote>
E. coli, one C. sakazakii and one P. aeruginosa isolates from chicken and yellow-legged gulls (Table 1). Co-presence of ESBL-producing strains e.g. C. sakazakii and E. coli was observed in a yellow-legged gull sample. These latter were resistant to amoxicillin; amoxicillin–clavulanic acid, cefalotin, ceftriaxone and were positive for the bla TEM-1 gene. One of the E. coli isolate (B32 strain) carrying bla TEM-1 was resistant to gentamicin and doxycycline, but none of the seven aminoglycoside resistance genes investigated were detected in this isolate. Besides, as expected, all strains isolated on the LBJMR agar were resistant to colistin (MIC ≥ 4 µg/mL) and included bacteria naturally resistant to colistin (Table 1). Resistance to β-lactams including amoxicillin, amoxicillin-acid clavulanic, and cephalothin was observed in most of H. alvei strains. Interestingly, for two H. alvei strains, an atypical resistance phenotype was observed, i.e. they were resistant to cephalothin but susceptible to amoxicillin (Table 1). All positive qPCR for targeted genes from samples were confirmed by standard PCR from isolated strains by culture of the corresponded sample. All these isolates were susceptible to carbapenems, amikacin, trimethoprim–sulfamethoxazole, ciprofloxacin, doxycycline, and gentamycin. Overall, qPCR and standard PCR searches for resistance genes encoding for bla SHV, bla VEB, bla GES, bla PER, carbapenemases, and mobile colistin resistance genes were negative for all isolates.

Genotyping by MLST was performed for *E. coli* isolates and revealed five different sequence types (STs) including ST10, ST34, ST38, ST1652 and a new ST which has been defined as ST857. The *E. coli* ST10 and ST38 carried the *bla*<sub>CTX-M-15</sub> gene and were from yellow-legged gulls, whereas the *E. coli* ST34 belonging to the ST10 complex was isolated from a pigeon and also harbored a *bla*<sub>CTX-M-15</sub> gene. Finally, *E. coli* ST1652, isolated from a chicken, carried the *bla*<sub>TEM-1</sub> gene and the *E. coli* ST-new carried *bla*<sub>CTX-M-1</sub> and was from yellow-legged gull.

## Discussion

In this study, we report CTX-M and TEM β-lactamases in Gram-negative bacterial isolates from urban birds in Marseille. Unlike previous studies reporting a high prevalence of ESBL-producing bacteria in gulls [11, 20, 21] and chickens [3, 22, 23], we observe here, a low prevalence (1.47%) of ESBL-producing bacteria in chickens. This is probably due to that they are domestic chickens that have never received food additives or growth factors and live only in non-livestock parks. Here, we isolated a β-lactamase-producing *P. aeruginosa* from a chicken which was susceptible to amoxicillin–clavulanic acid and harbored the *bla* TEM-1 gene only. With respect to the literature, *P. aeruginosa* is considered as one of the leading causes of infections in livestock and companion animals

and *P. aeruginosa* isolates producing ESBL genes, such as *bla* PER-1, *bla* CTX-M, *bla* SHV and *bla* TEM were reported in camel meat with a high prevalence [24]. Moreover, ESBL-producing *P. aeruginosa* isolates have also been observed from clinical settings, poultry, cattle and vegetables [25].

Interestingly, there was a simultaneous presence of ESBL-producing C. sakazakii and E. coli in a yellow-legged gull. In 2015, a study conducted by Zurfluh et al. [26] reported the presence of C. sakazakii producing ESBL SHV-2 type in fresh vegetables imported to Switzerland from the Dominican Republic, India, Thailand and Vietnam. It should be noted that C. sakazakii is an opportunistic Gram-negative, pathogenic bacterium with no spore formation, sometimes associated with sporadic cases or brief outbreaks of sepsis, meningitis, encephalitis, necrotizing enterocolitis and bacteremia in adults and newborns preterm neonates [27]. In many cases, the reservoir of C. sakazakii is unknown, and there are increasing reports that powdered infant formula is the source of C. sakazakii infections [28]. Our isolate was resistant to cephalothin as described by Xu et al. [29] who reported Cronobacter spp. isolates resistant to cephalothin in ready-to-eat foods and the potential risk of consumer contamination.

*Hafnia alvei*, an anaerobic Gram-negative bacterium, was isolated from all species birds investigated here. They are often present in the environment and a part of the animal gut [30]. This bacterium is rarely isolated from humans but can sometimes be an opportunistic pathogen responsible for nosocomial infections [30, 31]. All *H. alvei* isolated from birds in this study were resistant to cephalothin and colistin, which is a natural resistance [31]. However, most of these isolates were also resistant to amoxicillin and amoxicillin–clavulanic acid but susceptible to ceftriaxone, which is similar to a study conducted by Angela et al. [32] on broad spectrum cephalosporin-resistant *Enterobacteriaceae* isolated from humans.

Except the ST857 new sequence type identified here, the ST10, ST38, ST34 and ST1652 ESBL-producing E. coli described here are widely reported in humans, animals and in the environment (waters and wild birds) [6, 33–35]. Therefore, our study is consistent with previous studies in which human and animal isolates shared mainly identical STs, suggesting a possible transmission. In France, very few studies have been conducted on urban birds and our study is the first to investigate antibiotic-resistant bacteria in parks and beaches. We observed that ESBL-producing *Enterobacteriaceae* were more frequently isolated from beach gulls than chickens and pigeons as reported by Stedt et al. [8]. They reported that the ESBL-producing bacteria were frequently detected in gulls (906 ESBLs from a
collection of 3158 samples, 28.7%) with significant vari-
ations in prevalence rates between countries. We also
observed that the STs isolated in our study were com-
pletely different from the STs found in yellow-legged
gull in the other sites of south of France [7]. Pets, such
as dogs and cats, are potential sources of transmission
of antibiotic-resistant bacteria due to their close con-
tact with humans and the intensive use of antimicro-
bial agents in humans and in the domestic animals. It
should be noted that parks are open to dogs that are
suspected of being a source of resistance genes. Indeed,
the spread of resistance can occur directly or indirectly,
through feeding, water and animal waste disposal in
farm fields. Therefore, our study supports the idea that
the beaches can be considered as sources of antibiotic-
resistant bacteria.

**Conclusion**

Urban birds can be vectors for the transmission of ESBL-
producing bacteria between human and animals. It may
be due to their close contact with human activities, in
addition to the role of parks and beaches as a meeting
place for domestic and human animals, urban birds and
environmental waters. To the best of our knowledge, we
report here, for the first time, the presence of antibiotic-
resistant bacteria from birds, especially yellow-legged
gulls and chickens, in Marseille's parks and beaches. Fur-
ther works such as whole genome sequencing of such
bacteria from birds of different regions would be inter-
esting to investigate how such bacteria are circulating via
urban birds.

**Supplementary information**

**Supplementary information** accompanies this paper at https://doi.
org/10.1186/s13028-019-0486-9.

**Additional file 1.** Protein alignment of identified β-lactamase enzymes from bird samples. **A)** Sequence alignment of TEM-1 proteins compared with reference TEM-1 protein (JQ735917) retrieved from the ARG-ANNOT database. **B)** Sequence alignment of CTX-M proteins compared with reference proteins of CTX-M-15 (JQ686199) and CTX-M-1 (X92506). All sequenced *bla*~TEM-1~ genes (814-bp) exhibited 99.61% aa similarity with the TEM-1 reference sequence. *bla*~CTX-M-15~ and *bla*~CTX-M-1~ genes (855-bp) exhibited 100% aa similarity with CTX-M-15 and CTX-M-1 respectively.

**Additional file 2.** Sampling details according to the investigated locations.

**Acknowledgements**

The authors thank CookieTrad for proofreading the manuscript.

**Authors' contributions**

J-MR and SMD conceived and designed the study. EPN collected the samples, analyzed and interpreted data. WC, TL, and SC contribute to the analyses. IP performed corrections and critical revisions. EPN, SMD, and J-MR drafted the manuscript and/or critical revisions. All authors read and approved the final manuscript.

**Funding**

This work was supported by the French Government under the «Investissements d'avenir» (reference: Mediterranee Infection 10-IAHU-03).

**Availability of data and materials**

Not applicable

**Ethics approval and consent to participate**

Not applicable.

**Consent for publication**

Not applicable.

**Competing interests**

The authors declare that they have no competing interests.

**Author details**

¹ IRD, APHM, MEPHI, IHU-Mediterranee Infection, Aix-Marseille Univ, 19-21 bd Jean Moulin, 13005 Marseille, France. ² IHU-Mediterranee Infection, Marseille, France.

Received: 19 February 2019 Accepted: 22 October 2019

Published online: 31 October 2019

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