Background
Florfenicol, which is only used to treat animal infections, is a derivative of chloramphenicol that is active against chloramphenicol-resistant isolates [
1]. Resistance to chloramphenicol occurs mainly through the production of inactivating enzymes called chloramphenicol acetyl transferases (CATs) [
2] and chloramphenicol exporters, such as CmlA [
3]. Over the past decade, most reports have demonstrated that the bacteria causing animal respiratory diseases show high resistance levels to chloramphenicol but are susceptible to florfenicol [
4]. However, the resistance levels and number of bacteria that are resistant to florfenicol have increased due to the widespread use of florfenicol in the treatment of animal diseases [
5‐
7]. A study on 1001 bacterial isolates showed that the resistance rates for trimethoprim/sulfamethoxazole and tetracycline were 3.0% and 14.7% in
Actinobacillus pleuropneumoniae and 6.0% and 81.8% in
S. suis, respectively, while the resistance rate for florfenicol was < 1% for all strains [
8]. Other reports have cited different resistance rates. In Australia, 2.0% and 6.0% of
A. pleuropneumoniae and
Pasteurella multocida strains isolated from pig respiratory infections were resistant to florfenicol, respectively [
9]. The resistance rate of
E. coli strains from canine urinary tract infections to florfenicol was higher than that of other pathogens: 31.6% (36/114) [
5].
The first florfenicol resistance gene,
pp-flo (renamed
flo), was identified on a plasmid in the fish pathogen
Photobacterium damselae subsp.
piscicida in 1996 [
10]. The
floR gene is closely related (97% identity) to the
flo gene [
11], and their proteins share 47% amino acid sequence identity with the CmlA protein. The
floR gene was first reported in 1999 on the chromosome of the worldwide epidemic strain
Salmonella enterica serovar Typhimurium DT104 [
11]. The primary source of human DT104 infections was thought to be animal populations, with both direct contact and foodborne modes of transmission [
12]. The IncC plasmid R55, which was initially described to be capable of conferring non-enzymatic chloramphenicol resistance in the 1970s, was then identified in
Klebsiella pneumoniae [
13]. Currently, nine florfenicol resistance genes [
floR,
floRv,
floSt,
fexA,
fexB,
pexA,
cfr, optrA and
estDL136] have been identified. With the exception of
cfr and
estDL136, which encode a 23S rRNA methyltransferase and a hydrolase, respectively, all of the genes encode exporters [
14‐
18]. The
floR gene and its analogs have mainly been identified in gram-negative bacteria, whereas the other resistance genes have mainly been detected in gram-positive bacteria [
15‐
17].
Similar to other resistance genes,
floR has been identified on both chromosomes and plasmids and has often been associated with mobile genetic elements and genomic islands [
19,
20]. Mobile genetic elements enable translocation of the
floR gene between DNA molecules, such as chromosomes and plasmids. A plasmid carrying the
floR gene can spread among bacteria of the same and different species or genera via conjugation or transformation, thereby disseminating resistance [
21]. Bacteria generally obtain multiple resistance genes through the horizontal transfer of plasmids carrying resistance genes [
22].
K. pneumoniae, which is a member of the
Enterobacteriaceae, is an opportunistic pathogen for both animals and humans. This bacterium is pervasive in the natural environment and benignly colonizes the gastrointestinal tracts of healthy humans and animals. However, the bacterium is also capable of causing a wide range of diseases in humans and different animal species [
23].
K. pneumoniae strains are a common cause of health-care associated infections including pneumonia, urinary tract infections (UTIs), and bloodstream infections for critically ill and immunocompromised patients. These strains also infect healthy people in community settings, causing severe infections including pyogenic liver abscess, endophthalmitis, and meningitis [
24]. For example, in animals,
K. pneumoniae strains are well documented to cause mastitis and wounds in cattle [
25]; endometritis, cystitis, and liver abscess in horses; tracheitis and wounds in birds; cystitis, phlebitis and otitis externa in dogs; and cystitis in cats [
26].
K. pneumoniae has also been associated with classical foodborne disease outbreaks [
19]. Notably, the prevalence of antibiotic resistance is increasing among
Enterobacteriaceae, including
K. pneumoniae [
23,
27]. In this study, we used multiple genetic approaches to investigate the
floR gene in
K. pneumoniae isolates of human origin and to further demonstrate the potential transmission of this resistance determinant between animal and human pathogens.
Discussion
In this study, we found that among all the clinical
K. pneumoniae isolates detected, 20.42% (67/328) were resistant to florfenicol, of which 7.01% (23/328) carried the
floR gene, but 13.41% (44/328) were free of the
floR gene. A similar report demonstrated a
floR gene positivity rate of only 21.8% (26/119) among 119 florfenicol-resistant gram-negative bacilli from several freshwater Chilean salmon farms [
42]. Our MIC results for the 328 strains demonstrated that the
floR gene played a key role in the resistance of these bacteria to florfenicol. The
floR-positive strains had a much higher resistance rate (23/23, 100%) and much higher MIC values for florfenicol (22/23, 95.65% with MIC values ≥512 μg/mL) than the
floR-negative strains, which had a resistance rate of 14.43% (44/305) with only 1.64% (5/305) of the strains having MIC values ≥512 μg/mL. At present, of the nine florfenicol resistance genes
, the
floR gene is the only known florfenicol resistance gene that has been identified in
K. pneumoniae strains of either human or animal origin [
43]. Five genes (
fexA,
fexB,
pexA,
optrA and
cfr) were mainly identified in gram-positive bacteria [
15‐
17]. The
cfr gene has also been occasionally identified in
E. coli or
Proteus vulgaris [
44,
45] and
fexA and
pexA were once identified in
E. coli [
44]. The other three genes have only been identified in certain gram-negative bacteria (
floRv in
Stenotrophomonas maltophilia [
46],
floSt in
Salmonella [
47] and
estDL136 in
E.coli [
44]). We hypothesize that other mechanisms, such as exporters and enzymes, in addition to the known florfenicol resistance genes, may also be responsible for florfenicol resistance in gram-negative bacteria including
K. pneumoniae.
The
floR genes were located on both chromosomes and plasmids amidst various mobile genetic elements, indicating that horizontal transfer of the
floR gene occurred among bacteria of different species. The
floR gene was identified first on the chromosome of
S. typhimurium DT104 (
Salmonella typhimurium DT104) and then on a plasmid of
E. coli isolate BN10660 [
48] and was also identified on the IncC plasmid R55 harbored by
K. pneumoniae [
13] and on other sources [
17,
43]. In
S. typhimurium DT104, the
floR gene was included in a 12.5-kb region with multiple resistance genes. The
tetR and
tetA tetracycline resistance genes were located downstream of the
floR gene and were flanked by two integrons. One integron contained an
aadA2 gene and an incomplete
sulI resistance gene, and the other harbored a β-lactamase gene and a complete
sulI gene [
49]. In pKP18–125, the downstream region was a class 1 integron that contained 5 resistance genes (
acc(6′),
arr2, etc.) and was different from the 12.5-kb region of the
S. typhimurium DT104 chromosome. Interestingly, the sequence most similar to the
floR-containing fragment on pKP18–125 from a clinical
Klebsiella pneumoniae isolate was located on pEC012 (KT282968), a plasmid from an
E. coli strain isolated from a chicken [
50]. This finding suggests that horizontal transfer of the
floR-containing fragment occurred between bacteria of animal and human origins.
Our PFGE analysis revealed that two
floR-positive strains (KP5 and KP6) had similar PFGE profiles. They were isolated from the same sample type (sputum) but were found in different hospitalized patients during different time periods. Some
K. pneumoniae strains carrying resistance genes were previously reported to have caused outbreaks in European countries, indicating the potential risk of the spread of resistance genes through bacterial outbreaks, especially those caused by bacteria with resistance plasmids [
51]. Although the relationship between the two strains carrying
floR is still in question, effort should be made to avoid any pathogen outbreaks in hospital environments.