Following systemic administration, ceftiofur and cefquinome are mainly excreted in urine with a limited portion, excreted in faeces (EMEA/MRL/005/95, Summary of Product Characteristics, Annex 1; Naxcel 100 mg/mL). Information on what that means in terms of active concentrations over time in intestinal contents is not available in sources in the public domain. Such information is essential to evaluate the exposure of the gastro-intestinal microbiota of the target animal to the parent drug or active metabolites (CVMP/VICH GL 27; Guidance on the preapproval information for registration of new veterinary medicinal products for food producing animals with respect to antimicrobial resistance, CVMP/VICH/644/01-final).
Influence of cephalosporin use on occurrence of MRSA
As MRSA are resistant to all beta-lactams, use of any substance in that group may provide selective pressure. In human medicine, use of cephalosporins, other beta-lactams, fluoroquinolones and glycopeptides have been shown by metanalysis to be associated with an increased risk of acquisition of MRSA (Tacconelli et al., 2008). In view of the increasing occurrence of MRSA in animals, the risk associated with use of substances with a potential to select for MRSA-colonization of animals should be further examined. The potential influence of the use of products formulated as ‘long acting’, with long excretion times deserve special attention, as the time when concentrations are close to the MIC of intestinal and skin microbiota can be long. This document is focused on resistance with particular relevance for the third and fourth generation cephalosporins rather than all cephalosporins and other penicillinase stable beta-lactams. Emphasis will, therefore be on resistance in Gram-negative enteric bacteria and MRSA will not be further discussed in detail.
Influence of cephalosporin use on the evolution of genes encoding beta-lactamases
The beta-lactamases TEM-1 and SHV-1 are common in bacteria from various animals. These enzymes do not confer resistance but mutations in the genes encoding these enzymes lead to structural changes that can extend or alter the substrate specificity (Gniadkowski, 2008). Similarly, mutations in the genes encoding AmpC-type enzymes can give rise to extended spectrum AmpC with activity also against 4th generation cephalosporins (Ahmed & Shimamoto, 2008; Le Turnier et al., 2009; Mammeri et al., 2007, 2008; Wachino et al., 2006).
The evolution of ESBLs has been attributed to the selective pressure exerted by use of higher generation cephalosporins (Medeiros, 1997). There are a number of studies in human clinical settings in support of that (Gniadkowski, 2008). Blásquez et al. (2000) have suggested a broader view: that in vivo evolution of ESBLs is driven by the constant fluctuating pressure of various beta-lactams, including also penicillins and first generation cephalosporins. This may explain why many of the enzymes generated in vitro never occur naturally – only ESBLs with a truly broad-based resistance would survive and be selected for in an environment where different beta-lactams are used.
Current knowledge on use of cephalosporins as a driver of the evolution of ESBLs and AmpCs is based on laboratory studies and studies in human clinical settings. It is probable that the general principle applies also to animal production thus the use of third and fourth generation cephalosporins in animal populations, and possibly also of other beta-lactams, may favour the evolution of beta-lactamases in exposed bacterial populations.
Influence of cephalosporin use on selection and amplification of genes encoding beta-lactamases
Use of third generation cephalosporins is a recognized risk factor for ESBL colonization of patients in the human hospitals (Asensio et al., 1996;Quale et al., 2002; Saurina et al., 2000; Urbanek et al., 2007). Several authors have suggested that the use of ceftiofur in cattle and turkeys may have contributed to the spread in Salmonella in North America, of plasmid mediated AmpC-type beta-lactamases (Allen & Poppe, 2002; Dunne et al., 2000; Fey et al., 2000; White et al., 2001; Winokur et al., 2000). Until recently, there have been no specific studies on the influence of the use of third generation cephalosporins on resistance in Enterobacteriae in food producing animals.
In an experimental study, administration of a single dose of ceftiofur to turkey poults without detectable ceftiofur-resistant strains did not result in the emergence of resistant strains (Poppe et al., 2005). In a parallel experiment, the poults were dosed both with susceptible S. Newport and with E. coli carrying a large plasmid encoding AmpC-type beta-lactamases. The plasmid was readily transferred in the intestine to the Salmonella strain and also to a serotype of E. coli different from the donor in absence of any selective pressure. The experiment did not include a group receiving both antimicrobials and bacteria carrying resistance; hence the influence of ceftiofur on transfer and shedding of bacteria carrying resistance genes was not evaluated.
In a small experimental study, pigs were injected with ceftiofur, cefquinome or amoxicillin once daily for 3 days (Cavaco et al., 2008). Untreated animals served as controls. Almost all animals were positive for E. coli with CTX-M-1 before the start of the experiment. Animals were also inoculated with a CTX-M-1 producing strain of E. coli before the start of treatments. Significantly higher counts of cefotaxime-resistant coliforms were observed in all treated groups compared with controls for up to 22 days after the end of treatment. The cephalosporins had a more pronounced effect than amoxicillin.
Tragesser et al. (2006) studied the occurrence of ceftriaxone-resistant E. coli in dairy herds in Ohio, USA and linked the results to information on use of ceftiofur in the studied herds. Most of the isolates that showed reduced susceptibility to ceftriaxone carried a plasmid coding for CMY-2. Such isolates were recovered from at least one of the sampled cows in 10 of the 12 herds reporting use of ceftiofur, and in two of the seven herds reporting non-use (odds ratio 25, P = 0.01). The mean within-herd prevalence was 40% for herds reporting use of ceftiofur, compared with 9% for those reporting non-use. There was neither association at individual cow-level nor a linear relation between within-herd prevalence and treatment frequency. There was no attempt to analyse the influence of use of other antimicrobials on the farm. All CMY-2-producing isolates of E. coli were co-resistant to streptomycin, sulphonamides and tetracycline, and in addition, commonly also to gentamicin, kanamycin and trimethoprim–sulphonamides. Co-selection by other antimicrobials as well as management factors could account for the lack of linear relation between within-herd prevalence and use of ceftiofur.
In a study from Denmark, pigs in farms using and not using ceftiofur were sampled (Jorgensen et al., 2007). Escherichia coli with reduced susceptibility to third generation cephalosporins was demonstrated in 69 of 200 sampled pigs (five of 10 farms) but only in three of 200 animals in control farms (one of 10 farms). The difference was statistically significant (P = 0.02). Production of ESBL (CTX-M-1) was demonstrated in 19 isolates from two of the ceftiofur-using farms (not statistically different from farms not using ceftiofur). The study did not examine other drug-use practices in the farms.
Lowrance et al. (2007) studied the influence of administration of ceftiofur crystalline free acid (‘long acting’) to steers. Ceftiofur was administered subcutaneously to different cohorts at 6.6, 4.4 (single doses) and at 6.6 mg/kg thrice with 6 days interval). Untreated steers served as controls. Ceftiofur-resistant faecal E.coli were present at the start of the study, and administration of ceftiofur was associated with an increase in the proportion of E. coli resistant to ceftiofur during treatment in all treated groups. No changes in proportion of resistant isolates recovered using nonselective techniques were observed in co-mingled control animals. Almost all resistant isolates were co-resistant to at least chloramphenicol, streptomycin, sulphonamides and tetracyclines, a pattern associated with a multiresistance plasmid described in AmpC-producing Salmonella and E. coli (Winokur et al., 2001).
The influence of general use of antimicrobials on antimicrobial resistance in bacteria from calves in the US was studied in a field trial (Berge et al., 2006). Individual treatments transiently increased the shedding of multiply resistant E. coli compared with nontreated calves. The isolates were resistant to ceftiofur, which was the antimicrobial used for most of the individual treatments.
A longitudinal study over 5 months on healthy young calves on a dairy farm showed a persistent high prevalence (65–100%) of calves shedding ceftiofur-resistant, CMY-2 producing, E. coli (Donaldson et al., 2006). The isolates were all multi resistant and belonged to 59 clonal types. The farm reported use of various antimicrobials including ceftiofur but kept no individual records; thus no attempt to correlate use with resistance could be made.
The persistence of ESBLs of CTX-M type on a dairy farm in the absence of use of cephalosporins and other beta-lactams has been documented from the UK (Liebana et al., 2006). All use of beta-lactam antimicrobials, apart from intramammary use of cefquinome, was stopped during the study period in an attempt to remove the selective pressure. The prevalence of animals shedding CTX-M positive E. coli remained high over 6 months. As in studies on CMY-2, there was a diversity of clones but an almost complete predominance of one plasmid type carrying a gene encoding CTX-M and in addition, streptomycin resistance. Occurrence of E. coli producing ESBL and CMY in the apparent absence of use of cephalosporins has also been reported in broiler (Smet et al., 2008; MARAN 2005).
It has been argued that active concentrations of ceftiofur in the intestines of treated animals are very low, and that the substance is rapidly metabolized by the intestinal microbiota (Hornish & Kotarski, 2002). The studies quoted above show that the concentrations are sufficient to select for E. coli with resistance to third generation cephalosporins. The lack of clonality of resistant isolates reported in several studies clearly indicates horizontal dissemination of resistance genes.
Co-selection of resistance in Enterobacteriaceae by noncephalosporin antimicrobials
As discussed above, ESBL- or AmpC-producing bacteria are often also resistant to multiple other antimicrobials. In most cases, the genes encoding these unrelated resistance traits are linked on the same plasmid or transferable genetic element as the ESBLs. Many of the antimicrobials in question are used in veterinary medicine, e.g. neomycin, streptomycin, tetracycline, trimethoprim, sulphonamides and fluoroquinolones. A few of these substances are also used as growth promoters in some parts of the world. Of particular concern is the described association between CTX-M or AmpC encoding genes and plasmid-mediated quinolone resistance (Robicsek et al., 2006a). In human hospitals, use of fluoroquinolones has been identified as a risk factor for spread of CTX-M (Ben-Ami et al., 2006).
The frequent linkage of resistance genes implies that once the ESBL- or CMY-encoding genes have entered a bacterial population in a production unit, a broad range of antimicrobials, including beta-lactams such as amoxicillin, but also of structurally unrelated antimicrobials can favour their selection and spread between animals and between bacterial strains (co-selection). In the Netherlands, Salmonella and E. coli-producing ESBL have emerged and increased in prevalence in poultry without prior use of cephalosporins (MARAN, 2005). Similarly, a high prevalence of ESBL- and CMY-producing E. coli has been reported in Belgian broiler flocks (Smet et al., 2008). It is probable that use of either beta-lactams such as amoxicillin or non-beta-lactam antimicrobials, have contributed to the observed increase.