Escherichia coli is a Gram-negative, facultative anaerobe bacterium of the Enterobacteriaceae family. Since E. coli is ubiquitous in the gastrointestinal tract of warm-blooded animals, it has been extensively used to monitor AMR in food animals (including poultry) (18, 19). In addition, some E. coli strains hosted by poultry are potential source of AMR genes that may transmit to humans (20, 21).

Certain E. coli strains, designated as “avian pathogenic E. coli” (APEC) are causative agents of colibacillosis, one of the principal causes of morbidity and mortality in poultry worldwide (22). Only studies investigating APEC strains are included in this review, therefore excluding studies on E. coli from chicken enteric samples (23, 24).

A total of 12 publications investigated phenotypic resistance in a total of 1,331 APEC isolates from diseased chickens, from Asia (25–29), Africa (30–32), the United States (33, 34), Spain (35), and Brazil (36) (Table 1). All studies included APEC isolates from the chicken species, except one study that, in addition, included isolates from ducks and geese (26). All studies were carried out using the disk diffusion test, except two where isolates were tested using microbroth dilution, and one that used the broth dilution test (Table 1). Two studies reported the MIC distribution of investigated strains (33). A study on 100 APEC strains from Iran reported 99% resistant strains against colistin using the disk diffusion test (28), but it is not clear what breakpoints were used. In addition, colistin resistance cannot be reliably estimated using disk sensitivity tests (37).

Results of phenotypic resistance in APEC are presented in Table S1 in Supplementary Material. Resistance levels of strains were: ampicillin (median 82.0%; IQR 59.0–95.7%), amoxicillin (80.0%; IQR 43.0–93.0%), ceftiofur (8.5%; IQR 5.4–52.9%), streptomycin (69.0%; IQR 46.0–86.0%), gentamicin (30.9%; IQR 18.5–43.9%), kanamycin (31.0%, IQR 23.5–79.0%), chloramphenicol (63.5%, IQR 36.9–79.5%), florfenicol (20.9%; IQR 9.4–41.0%), tetracycline (91.0%; IQR 53.7–96.7%), co-trimoxazole (60.8%; IQR 34.0–85.6%), nalidixic acid (83.0%, IQR 77.0–88.0%), ciprofloxacin (67.0%; IQR 28.3–86.0%), and enrofloxacin (32.0% IQR 5.1–76.0%).

A study from China identified floR, cmlA, cat1, cat2, and cat3 (genes associated with florfenicol and chloramphenicol resistance) among APEC strains (25). In another study from the same country, the presence of class I integrons on isolates from the same country was strongly correlated with multi-drug resistance (93.3% MDR strains were positive for class 1 integron, compared with 12.5% among non-MDR strains) (26).

In a study from Egypt integrons (mostly class 1) were detected in 29.3% isolates, and were associated with the presence of genes encoding for resistance to trimethoprim (dfrA1, dfrA5, dfrA7, dfrA12), streptomycin/spectinomycin (aadA1, aadA2, aadA5, aadA23), and streptothricin (sat2). Other, non-integron-associated resistance genes, included tetracycline (tetA and tetB), ampicillin (bla_TEM), chloramphenicol (cat1), kanamycin (aphA1), and sulfonamide (sul1 and sul2). The S83L mutation in the gyrA gene (present in 23.2% isolates) was the most frequently genetic determinant of quinolone resistance, followed by qnrA, qnrB, and qnrS genes (31). A previous study on 73 APEC strains from the same country (of which 67.0% were nalidixic resistance, 15.1% ciprofloxacin resistance), plasmid-mediated quinolone resistance genes qnrA1, qnrB2, qnrS1 were found in 64.0% isolates, and the fluoroquinolone-modifying acetyltransferase gene (aac(6_)-Ib-cr) in 7.0% isolates (32). However, the study did not investigate quinolone resistance encoded by genetic mutations.

A study on 116 APEC isolates from broilers in Egypt showed a remarkably high percentage of ESBL-producing strains (58.6%). The bla_TEM and bla_CTX−M−1 genes were the most prevalent genes in these strains (31). In a study from Spain of 11 cephalosporin resistant isolates, 6 contained bla_CTX-M-14, 2 bla_SHV-12, 2 bla_CMY-2, and 1 bla_SHV-2 (35).

A recent study on a large collection (980) of APEC isolates from several countries identified the plasmid-mediated mcr-1 colistin resistance gene in 8 isolates from China (of 31 tested) and 4 from Egypt (of 20 tested). Most such strains were multi-resistance to 10 or more antimicrobials (38).

A study on APEC isolates from Jordan investigated the most effective synergistic effects of combinations of 11 antimicrobials by calculation of a fractional inhibitory concentration index of checkerboard titrations. The combinations of amoxicillin–clavulanic acid, ciprofloxacin–fosfomycin, oxytetracycline–erythromycin, oxytetracycline–florfenicol, amoxicillin–gentamicin, oxytetracycline–spectinomycin, and spectinomycin–erythromycin were the most effective in vitro (27).

Salmonella Pullorum/Gallinarum are biovars within the genus S. enterica subspecies enterica within the family Enterobacteriaceae. They are the etiological agents of pullorum disease (S. Pullorum) and fowl typhoid (S. Gallinarum), two septicemic diseases widely common in much of the world, though they have been eradicated from commercial poultry operations in many developed countries (39, 40).

Eight publications investigated phenotypic resistance in a total of 780 S. Pullorum/Gallinarum isolates from Korea (four publications) (41–44), India (two) (45, 46), Brazil (one) (47), and China (one) (48) (Table 2). All studies used the disk diffusion test, except one study where agar dilution was used (44), and one where both tests were used (41).

Overall levels of phenotypic resistance were: ampicillin (median 13.0%; IQR 4.1–38.1%), amoxicillin plus clavulanic acid (0%; IQR 0–0%), cefotaxime (0%; IQR 0–1.2%), streptomycin (27.0%; IQR 0–58.0%), gentamicin (2.6%; 0–43.4%), chloramphenicol (0%; IQR 0–0%), tetracycline (11.2%; IQR 0–37.7%), co-trimoxazole (0%; IQR 0–1%), nalidixic acid (69.0%; IQR 38.2–86.6%), ciprofloxacin (2.0%; 0–33.0%), enrofloxacin (2.8%; IQR 0–10.5%).

A study from Korea reported an increase over time in phenotypic resistance among S. Gallinarum isolates: whereas in 1995 all isolates were fully susceptible to 12 antimicrobials, except for tetracyclines (>83% resistance), by 2001, levels of resistance were: ampicillin (87.0%), gentamicin (56.6%), kanamycin (30.4%), enrofloxacin (93.5%), ciprofloxacin (89.1%), norfloxacin (47.5%), and ofloxacin (17.4%) (41). Over the same period, the MIC range for enrofloxacin, ciprofloxacin, norfloxacin, ofloxacin also increased considerably, in parallel with an increase in the rate of mutations of the gyrA (from 5.6 to 89.1%) (42).

A further study from the same country unexpectedly identified S. Gallinarum in table eggs from healthy chicken layer flocks (43). Surprisingly, isolates were pan-susceptible for most antimicrobials except for streptomycin (88.5% were intermediate resistance).

A study of 42 quinolone resistant strains identified the substitution of a Ser to a Phe or Tyr at position 83 in the gyrA gene among 71.0% of isolates. The study identified three different class 1 integrons among 57 sulfonamide resistant strains, containing resistance genes aadA (52.6%), aadB (12.3%), or aadB-aadA. In addition, isolates harboring the integron containing aadB-aadA displayed resistance to aminoglycosides, as well as increased resistance to fluoroquinolones. As in the case of E. coli strains, it is suspected that integrons are largely responsible for multi-drug resistance; clonal expansion and horizontal gene transfer may have contributed to the spread of AMR integrons in these organisms (49).

A study of 337 S. Pullorum strains from China showed a consistent increase in resistance between 1962 and 2010. Resistance levels against 11 of 16 antimicrobials tested was significantly greater among int1(+) than int1(−) isolates, and resistance levels to cefamandole, trimethoprim and co-trimoxazole were significantly higher for biofilm-positive types compared with the biofilm-negative groups (48). Recently, full genome sequencing of a multi-drug resistant S. Pullorum isolate from China has been published, and included two prophages, the ST104 and prophage-4 (Fels2) previously found in E. coli (50).

Pasteurella multocida is a Gram-negative, non-motile, facultative anaerobic bacterium of the Pasteurellaceae family. It is the causative agent of fowl cholera, a disease that often manifests as acute fatal septicemia in adult birds, although chronic, and asymptomatic infections also occur (51).

A total of eight publications have investigated phenotypic resistance in P. multocida isolates, including studies from the United States (33, 52), Brazil (53, 54), India (55), Indonesia (56), Hungary (57), and Egypt (58) (Table 3). Four studies investigated isolates originating exclusively from the chicken species, and the other four included isolates from ducks, geese, Muscovy ducks, pheasants, and quails, in addition to chicken isolates. Five studies used disk diffusion, and three the broth microdilution technique.

In total, 617 isolates were tested in such studies. Overall levels of phenotypic resistance were: ampicillin (median 2.3%; IQR 0.6–13.5%), gentamicin (4.3%; IQR 1.8–11.1%), erythromycin (18.0%; IQR 2.7–64.1%), florfenicol (0.6%; IQR 0–1.6%), tetracycline (13.8%; IQR 7.6–40.0%), co-trimoxazole (10.8%; IQR 0–20.0%), and enrofloxacin (4.7%; IQR 1.0–22.0%). A study on 120 isolates from poultry in India showed 100% resistance against sulfadiazine, a drug most often used in the field to treat fowl cholera in that country. Only resistance against chloramphenicol, ciprofloxacin, norfloxacin, enrofloxacin, gentamicin, and lincomycin, was observed in <10% isolates, remaining the only effective therapeutic alternatives (55). However, the authors did not provide the interpretation criteria, other than “provided by the disk manufacturer.” In another study of 56 poultry isolates from Brazil, levels of resistance were highest for sulfonamides (sulfaquinoxaline) (~77%); in contrast levels of resistance against β-lactams (amoxicillin, ceftiofur), aminoglycosides (gentamicin), and macrolides (erythromycin) were <6%. In a study from the United States of 80 isolates, resistance was less than 7% against all antimicrobials. In comparison with E. coli and Salmonella isolates, P. multocida isolates from poultry were found to be much more susceptible to the antimicrobials tested (33).

Studies on isolates from pigs, cattle, and poultry in Europe have shown that resistance in P. multocida is generally mediated by small (4–7 kb size) plasmids (59, 60). A larger plasmid (pVM111) has also been shown to contain multiple genes conferring resistance against tetracyclines, sulfonamides, and streptomycin resistance (tetR-tet(H), sul2, and strA), supporting the hypothesis that the spread of resistance is due to horizontal transfer of plasmids rather than clonal dissemination. It is not known whether this explains the relatively lower prevalence of AMR in P. multocida compared with other Gram-negative bacteria (61).

Avibacterium paragallinarum (previously H. paragallinarum) is a capsulated, rod-shaped, Gram-negative facultative anaerobe bacterium of the Pasteurellaceae family. It is the etiological agent of infectious coryza, an acute disease of the upper respiratory tract of chickens worldwide (62).

A total of seven publications investigated phenotypic resistance in 143 A. paragallinarum isolates from diseased flocks in Asia (India, Thailand, Indonesia, Taiwan) (63–67), Africa (Uganda) (68), and the Americas (Mexico, Ecuador, Peru, Panama) (5). All studies used the disk diffusion test, except one where broth microdilution (64), and one where agar dilution (68) tests were used (Table 4). Initial MIC interpretative criteria (breakpoints) of resistance for A. paragallinarum were provided by Fales et al. (1986) and cited by Blackall (8) (Table 4). Overall levels of phenotypic resistance for the main antimicrobials tested were: ampicillin (median 38.9%; IQR 5.9–60%), neomycin (77.4%; IQR 56.2–100%), streptomycin (72.7%; IQR 62.1–88.9%), erythromycin (77.8%; IQR 69.8–86.3%), co-trimoxazole (44.1%; IQR 19.6–67.0%).

A comparison of results between the disk diffusion method (65) and the broth microdilution (66) on the same panel (18 isolates) from Thailand revealed important discrepancies in the interpretation of results, notably for ampicillin (33.3% disk diffusion vs. 5.6% broth microdilution), amoxicillin (27.8 vs. 0%), ceftiofur (27.8 vs. 5.6%), enrofloxacin (27.8 vs. 50.0%), and spectinomycin (11.1 vs. 50.0%).

A study on isolates from Latin American countries (5) showed the lowest level of resistance against co-trimoxazole (potentiated sulfonamide). However, the authors remind that sulfonamides should be administered with caution in poultry given their low safety margin and the presence of residues in meat and eggs for a relatively longer period (13).

A study of four A. paragallinarum isolates in Tanzania detected genes associated with streptomycin (strA), ampicillin (bla_TEM), tetracycline (tetC and tetA), and sulfamethoxazole (sul2) resistance (68). In a study of 18 isolates from Taiwan about 72% isolates contained plasmids pYMH5 and pA14 (64). Sequencing data indicated that pYMH5 encodes functional streptomycin-, sulfonamide-, kanamycin-, and neomycin-resistance genes (sul2, strA, mbeCy, aphA1).

Gallibacterium anatis biovar haemolytica is a Gram-negative bacterium of the Pasteurellaceae family. The organism is known to colonize the upper respiratory tract and lower reproductive tract of chickens, but also been experimentally shown to induce clinical infection (69). G. anatis has previously been misclassified as M. haemolytica, P. hemolytica, P. anatis, and Actinobacillus salpingitidis, but was recently classified as a new genus (Gallibacterium) (70). Surveillance data from the state of Mississippi (US) confirmed a progressive increase in confirmed cases of G. anatis from 2006 to 2011. By 2011, the annual number of confirmed cases of disease (28) was comparable with those of fowl cholera (32) (52). A total of three studies have investigated phenotypic resistance in G. anatis (34, 52, 71). However, in one of them, these isolates were identified as M. haemolytica (34). However, in the absence of specific breakpoints published, one study only indicated the mean inhibition zone of isolates, indicating that isolates showed maximum sensitivity to norfloxacin (32 mm) and minimum (16 mm) to erythromycin (71). Generally, levels of resistance were higher than those observed for P. multocida and A. paragallinarum (Table 5).

Ornithobacterium rhinotracheale is a Gram-negative, rod-shaped bacterium that causes respiratory disease in turkeys, chickens, and other avian species. It was first identified in turkeys in the 1990s (72). Establishing the antibiotic sensitivity of this pathogen is difficult because of its complex growth requirements. ORT is known to be often resistant to many antimicrobials, and therefore only isolates from wild birds are likely to display the highest degree of susceptibility; therefore, antimicrobial susceptibility results in these isolates have often been used to compare with those from poultry isolates (73).

Four studies investigated phenotypic resistance on ORT isolates from the Netherlands (74), Belgium (75), Hungary (76), and the Unites States (77) in a total of 600 isolates. The overall prevalence of resistance of such studies were: ampicillin (median 40.0%; IQR 11.3–100%), ceftiofur (63.0%; IQR 50.0–100%), tetracycline (21.0%; IQR 20.0–61.0%), co-trimoxazole (89.0%; IQR 25.0–97.0%), and enrofloxacin (70.8%; IQR 33.4–93.6%) (Table 6).

A study determined MICs for 10 antimicrobials of 10 Mexican ORT isolates alongside 10 previously characterized strains. MIC values greater than 128 mg/mL were recorded for gentamicin, fosfomycin, trimethoprim, sulfamethazine, sulfamerazine, sulfaquinoxaline, and sulfachloropyridazine were identified among isolates. Field reports from that country confirmed that the use of gentamicin or fosfomycin had no effect when used in therapy in infected flocks, and based on these results, the authors recommended that amoxicillin, enrofloxacin, or oxytetracycline as drugs of choice (78). A study from China reported that small-colony variants, had overall higher MICs levels compared with their wild-type counterparts. Differences were also found with regards to other phenotypic characteristics, but not in their genotype (79).

A study investigated the mechanisms of enrofloxacin resistance after experimental inoculation and treatment of turkey flocks, and found that mutations in gyrA commonly developed after a single treatment, and it was associated with an increase in MIC (increase in MIC from 0.03 to 0.25 mg/mL), among field isolates (80).

Bordetella avium is a Gram-negative, strictly aerobic bacterium, of the family Alcaligenaceae. It is the etiological agent of turkey coryza, a respiratory disease of economic importance to the turkey industry (81). In addition, the organism can however also colonize a range of wild and domestic birds (82, 83). In addition, B. avium organism is considered to be zoonotic, since it has been isolated from human patients with respiratory disease (84).

A total of three studies investigated phenotypic resistance in a total of 50 B. avium isolates, 1 by disk diffusion (from the United States) (34), 1 by broth microdilution (United States) (85), and 1 using both (Europe) (76) (Table 6). In all three studies, turkey isolates were investigated. However, one study also included chicken and pigeon isolates (76). Interpretation of results in both studies was based on criteria “for fastidious Gram-negative bacteria” (34, 76). However, in one study, the prevalence of resistance was determined in relation to the observed MICs for the antimicrobials tested (85).

A study on farmed cockatiel chicks affected with lockjaw syndrome (characterized by anorexia, sneezing, coughing, nasal discharge, and swollen infraorbital sinuses) investigated 10 isolates by disk diffusion. Isolates were sensitive to ampicillin, amoxicillin, penicillin, ceftiofur, enrofloxacin, norfloxacin, ciprofloxacin, erythromycin, florfenicol, and co-trimoxazole, whereas resistance to lincomycin and sulfadimethoxine were common to all of the isolates, and four strains showed resistance to tetracycline (86).

An experiment investigated transfer of a 12–13 kb plasmid (pRAM) coding for tetracycline and sulfonamide resistance to a receptor strain. Partial DNA sequence analysis of pRAM revealed two genes for conjugation, similar to P-type conjugative transfer ATPase, TrbB, and TrbC of Enterobacter aerogenes (85). There is lack of data on additional mechanisms of resistance of this poultry pathogen.

Clostridium perfringens is a Gram-positive, rod-shaped, anaerobic, spore-forming bacterium commonly found in the intestinal tract of poultry, animals, and the environment. Under certain conditions, the bacterium can multiply, causing necrotic enteritis, and cholangiohepatitis, two diseases that are responsible for heavy losses in the broiler and turkey industry worldwide (87).

A total of seven publications have investigated phenotypic resistance in 564 C. perfringens isolates from Belgium (88), Scandinavia (89), Egypt (90), Korea (91), Brazil (92), and Canada (93). All studies investigated chicken isolates, except one that also included isolates from turkey species (92).

Agar dilution and broth microdilution methods were used in three and two publications, respectively. In all studies, the MIC distribution of tested strains was provided. In additional to conventional antibacterial antimicrobials, a number of studies have investigated resistance against antimicrobials commonly used as growth promoters (bacitracin, avilamycin, virginiamycin) in addition to coccidiostats (i.e., salinomycin, monensin) that are also known to have activity against Clostridium spp. in the gut (94).

The calculated MIC50 levels for: erythromycin 2 µg/mL (IQR 2.0–5.0), tetracycline (8 µg/mL; IQR 4.5–8), bacitracin (8 µg/mL; IQR 1–128), avilamycin (0.25 µg/mL; IQR 0.25–2.0), naransin (0.25 8 µg/mL; 0.06–0.25), salinomycin 0.5 µg/mL (0.12–0.5), and monensin (0.63 µg/mL; 0.25–1.0). Susceptibility cut-offs were determined based on the observed distribution of MICs. In addition, two studies used disk diffusion methods. However, in one publication, interpretation guidelines were not provided (Table 7). Overall levels of resistance were: tetracycline (median 66.6%; IQR 41.8–70.7%), lincomycin (62.4%; IQR 33.6–81.6%), erythromycin (17.5%; IQR 0.6–100%), bacitracin (7.5%; IQR 3.0–56.0%), ampicillin (median 0%; IQR 0–3.5%), and florfenicol (0%; IQR 0–1.0%).

A study from Belgium on isolates collected during 2007 found high (60–70%) levels of resistance against lincomycin and tetracycline, but susceptibility to six other antimicrobials tested. However, the authors found no evidence of increases in the prevalence resistance against these antimicrobials compared with the earlier period 1980–2004 (95).

A study from Taiwan reported MIC50 values of erythromycin and lincomycin for C. perfringens isolated from intestinal samples with severe lesions were significantly higher compared with those with mild lesions (96). However, a study from Korea compared resistance patterns between isolates from healthy and sick flocks, and found no difference (91). Studies on C. perfringens isolates from Canadian chickens and turkeys had overall higher levels of resistance against bacitracin and virginiamycin compared with bovine and porcine isolates (92), but not for other antimicrobials tested.

Studies in Belgium and Scandinavia have identified tetP(B), tet(M), tetA(P), and tetB(P) genes among tetracycline resistant isolates (88, 89). Genes lnu(A) and lnu(B) genes associated with low-level resistance against lincomycin have identified in strains from Belgium (88).

Mycoplasma spp. are Mollicutes bacteria that lack a cell wall around their membrane. M. gallisepticum (MG) infection is particularly important as a cause of respiratory disease and decreased meat and egg production in chickens and turkeys worldwide. Other species such as M. synoviae (MS) M. meleagridis, and M. iowae can also cause disease in poultry (97).

Since Mycoplasma spp. are fastidious organisms, routine methods based on isolation and phenotypic testing of resistance are not practicable. Mycoplasma spp. are unaffected by many common antibiotics that target cell wall synthesis. Antimicrobials commonly used to treat Mycoplasma spp. infections include tetracyclines, macrolides (tylosin, tilmicosin), and more recently, fluoroquinolones (enrofloxacin, difloxacin), and pleuromutilins (tiamulin).

A total of five studies have determined MICs among a total of 145 MG and 43 MS field strains, all using broth microdilution. Studies were carried out in Israel (98, 99), Jordan (100), Iran (101), and Thailand (102). One study compared MIC results using broth microdilution, agar dilution, and E-test methods (98). MIC data were not converted into prevalence of resistance due to the lack of published standards. For those antimicrobials included in at least three studies, the median MIC50 values (and IQRs) were, in decreasing order: erythromycin (8.8 µg/mL; IQR 0.05–128), chlortetracycline (2.73 µg/mL; IQR 1.0–4.0), enrofloxacin (1.48 µg/mL; IQR 0.26–11.31), tylosin (0.125 µg/mL; IQR 0.015–0.33), and doxycycline (0.062 µg/mL; IQR 0.015–0.2).

In vitro studies involving passages in sub-inhibitory concentrations of antimicrobials have shown resistance to macrolides can be quickly acquired among poultry Mycoplasma spp., whereas resistance to enrofloxacin develops more gradually. No resistance to tiamulin or oxytetracycline could be evidenced in MG or MS after 10 passages, whereas M. iowae resistant mutants were obtained. Mycoplasma spp. mutants that became resistant to tylosin were also resistant to erythromycin, whereas mutants made resistant to erythromycin were not always resistant to tylosin (103).

A study on MG and MS isolated from chickens and turkeys in Israel collected during 2005–2006 indicated a reduction in susceptibility against fluoroquinolones (enrofloxacin and difloxacin) compared with archived strains (1997–2003) (98). Similarly, a study from Jordan compared MICs in isolates collected from 2004 to 2005 vs. strains collected during 2007–2008 confirmed a significant increase in MIC against 8 (erythromycin, tilmicosin, tylosin, ciprofloxacin, enrofloxacin, chlortetracycline, doxycycline) of 13 antimicrobials tested (100). A study on 20 MG isolates from Thailand where MG isolates were further characterized into groups (A, B, C, D, U) by random amplification of polymorphic DNA reported the lowest MICs for doxycycline, tiamulin, and tylosin among all tested drugs. Some MG isolates low-level resistant to josamycin and were resistant to enrofloxacin and erythromycin (102).

Tiamulin (pleuromutilin) has been found in general to be a useful drug in the treatment and control of Mycoplasma spp. infection. However, administering tiamulin to flocks medicated with ionophore antimicrobials is not recommended, since it may lead to toxicity (104).

Fluoroquinolone resistance in Mycoplasma spp. is of great concern, since enrofloxacin is often the drug of choice to treat infections in poultry. However, a study showed that treatment with enrofloxacin did not succeed in eradicating infection from flocks subjected to experimental infection (105).

Resistant mutants of MG were selected in vitro by passaging have been shown to be due to amino acid substitutions in the gyrA, gyrB, parC, and parE genes (106, 107). A study on 93 strains from several countries indicated that MG strains with substitutions in the quinolone resistance-determining regions (QRDRs) of both gyrA and parC are resistant to enrofloxacin, however in 10% strains with such substitutions did not show a clear correlation with the MIC. The authors concluded that this may limit the applicability of a gene-based assay to detect fluoroquinolone resistance in this avian pathogen (108).