Aminoglycoside acetyltransferase 3-IV (aacC4) and hygromycin B 4-I phosphotransferase (hphB) in bacteria isolated from human and animal sources.

Members of the family Enterobacteriaceae harboring an enzyme of the aminoglycoside acetyltransferase 3 class (AAC-3-IV) (apramycin and gentamicin resistance) and hygromycin B phosphotransferase 4 (HPH-4-I) (hygromycin B resistance) have been isolated from human clinical sources in Europe. A cluster of genes containing IS140, aacC4, and hphB was found in these strains. We demonstrate by Southern hybridization that this cluster is identical to the operon found in animals that also contains insertion sequences belonging to the ISO family. This provides another example of presumptive transfer of antibiotic resistance genes between bacteria of animal and human origin. Images

class (AAC-3-IV) (apramycin and gentamicin resistance) and hygromycin B phosphotransferase 4 (HIPH-4-I) (hygromycin B resistance) have been isolated from human clinical sources in Europe. A cluster of genes containing IS140, aacC4, and hphB was found in these strains. We demonstrate by Southern hybridization that this cluster is identical to the operon found in animals that also contains insertion sequences belonging to the IS6 family. This provides another example of presumptive transfer of antibiotic resistance genes between bacteria of animal and human origin.
Apramycin and hygromycin B are two members of the aminoglycoside family of antibiotics which have been used extensively for veterinary applications but have never been employed in human therapy. Gram-negative bacterial strains resistant to these antibiotics have been isolated from several different animal sources, and the associated resistance mechanisms have been characterized (1,4,9,13). Apramycin resistance is due to N acetylation by an enzyme of the aminoglycoside acetyltransferase 3 class (AAC-3-IV), and hygromycin inactivation is due to 0 phosphorylation by hygromycin B phosphotransferase 4 (HPH-4-I). The corresponding genes (aacC4 and hphB) have been cloned and sequenced (1,7,10); aacC4 and hphB form part of an antibiotic resistance gene operon, which is transcribed from a promoter 5' to the aacC4 gene (1,10).
AAC-3-IV, but not HPH-4-I, is capable of modifying a number of other aminoglycoside antibiotics, including gentamicin and tobramycin. Since the latter are used widely in human therapy and since bacterial resistance due to several different AAC-3 types has been generally reported, it might be anticipated that AAC-3-IV would be found in aminoglycoside-resistant isolates from humans. This has been noted recently in Europe (R.  675,1989). It was of interest to see whether the AAC-3-IV of such isolates was encoded by the same aacC4 as in isolates from animals, and more pertinently to determine whether or not * Corresponding author. the apramycin resistance gene in the human clinical isolates was associated with hphB in the same configuration as in veterinary isolates. We demonstrate by restriction analysis and Southern hybridization that this was the case, consistent with the notion that resistance gene transfer has taken place between bacterial populations in animals and humans.

MATERIALS AND METHODS
Strains. Plasmids pIP1508, pIP1509, and pIP1510 (Table 1) were described previously (2), had been isolated from Salmonella typhimurium or Escherichia coli in calf feces in France, and were a gift from E. Chaslus-Dancla (Institut National de la Recherche Agronomique, Tours, France). Plasmids pUZ7852, pUZ7874, pUZ6776, pUZ6734, and pUZ7343 were isolated from E. coli, Klebsiella pneumoniae, or Serratia marcescens in the blood or urine of patients in Madrid or Zaragoza, Spain. E. coli 86061324 of human origin was obtained from G. Miller (Schering-Plough). The plasmids from these strains encoded resistance for several aminoglycoside antibiotics (Table 2) and other antimicrobial agents (results not shown). The calves had been intensively treated with apramycin and gentamicin; some of the human patients (from whom K. pneumoniae 34 and E. coli 6734 were isolated) had been treated with gentamicin. Hygromycin B was not known to have been used in any instance.
The E. coli strain harboring plasmid pWP7b (see Fig. 3A) used for preparing the IS140 and aacC4 gene probes used for hybridization has been previously described (1). Plasmid pPM732 (unpublished data), a gift of P. Mazodier, is a multicopy derivative of pLG61 (7) containing the hphB gene from pJR225 (4).
Microbiological techniques. Enterobacterial strains were isolated on Drigalski or MacConkey medium, and an API system (API-System, La Balme les Grottes, France) was used to identify the strains. Antibiotic resistance was determined by the disk diffusion test on Mueller-Hinton agar.
MICs were determined on the same medium (5).
Assay for aminoglycoside-modifying enzymes. Crude bacterial extracts of recipient strains were prepared by sonication, and the enzymes were assayed by a phosphocellulose paperbinding assay (8).
DNA preparation and agarose gel electrophoresis. Plasmid DNA was prepared by alkaline lysis (11), except that the volume of culture used for the minipreparation was increased from 1.5 to 20 ml. Agarose gel electrophoresis was performed with 0.7% agarose in TAE buffer (11). It was found that the presence of 0.2% sodium dodecyl sulfate in the bromophenol blue dye buffer enhanced the penetration of undigested plasmid DNA in the agarose. The physiochemical basis of this phenomenon is not known.
Probe preparation. The probes used for Southern hybridization were intragenic fragments obtained from plasmid pPM732 (probe hphB) or pWP7b by digestion and cloning in the multicopy vector pUC18 or pUC19 (16) in order to obtain recombinant plasmid pDSA701 (probe IS140) or pDSA702 (probe aacC4). The probe for IS140 consisted of the 0.2kilobase (kb) PstI-SalI fragment of the IS140 element, the probe for aacC4 was a SacI 0.75-kb intragenic fragment, and the probe for hphB was an EcoRI-SacII 0.55-kb intragenic fragment. After digestion of pPM732 or the recombinant plasmids, these fragments were then isolated by electrophoresis on 8% acrylamide thin gels and subsequent elution by passive diffusion in 0.3 M sodium phosphate-0.1% sodium dodecyl sulfate buffer at 42°C.

RESULTS
Resistance to hygromycin B. All the apramycinand gentamicin-resistant strains, regardless of source, were also resistant to hygromycin B (Table 2). Resistance enzyme analyses showed that these strains harbor both aacC4 and hphB, according to their typical substrate profiles (reference 2 and results not shown). Table 3 shows the results for gentamicin Cla, apramycin, hygromycin B, and amikacin. Apramycin was modified only by acetylation and hygromycin B was modified only by phosphorylation, as previously reported (4, 10). Resistance plasmids. DNA was isolated from each strain after conjugation to E. coli (2), and the result of electrophoretic separation of fragments from the different plasmids after digestion with SalI and BglII is shown in Fig. 1A; it was known (1) that the aac-hph cluster in pWP7b is flanked by these two restriction sites. The sizes of the plasmids were pIP1509; lanes 4, pIP1510; lanes 5, pUZ7852 (several plasmids?); lanes 6, pUZ7874; lanes 7, pUZ6776; lanes 8, pUZ6734; lanes 9, pUZ7343; lanes 10, probe for IS140; lanes 11, probe for aacC4; lanes 12, probe for hphB (these three probes were deposited on the gel just before the end of the run); lanes 13, pWP7b (two extra restriction bands appear in comparison with lanes 16, probably because of an extra Sall site including a methylated thymine for which digestion was partial); lanes 14, pUZ7343 (the amount of DNA used was five times more than that used in lanes 9); lanes 15, 86061324 (several plasmids); lanes 16 estimated to be 40 kb for pIP1508 (2) and about 100 to 150 kb for the others; several of the strains contained more than one plasmid. The restriction enzyme digestion profiles were quite similar for plasmids of the five human strains, and several common fragments were also observed in pIP1510 and the plasmids from human isolates.
Hybridization studies. The BgIII-SalI digestions (Fig. 1) were transferred to filters and hybridized successively with the three probes, with complete stripping between each hybridization. The probe for IS140 identified a 2.9-kb fragment in all 10 plasmids from the various sources (Fig. 1B). It also hybridized to two additional DNA fragments of pIP1510, five of pUZ7343, and at least six of 86061324. Hybridization with the probes, for aacC4 and hphB ( Fig. 1C and D) identified the same 2.9-kb fragment in all the plasmids studied. Two extra hybridization bands were seen for 86061324, which suggested that in this strain three aacC4-hphB pairs were carried by three restriction endonuclease fragments of different sizes, probably because of rearrangements and insertions within the SalI-BglII restriction fragments.
To investigate the possible involvement of transposition, we used the IS140 probe to analyze pIP1510. Two filters were prepared, the first after PstI digestion ( Fig. 2A) and the second after Sall digestion (Fig. 2B). Hybridization with the IS140 probe indicated the same number of positively hybridizing fragments as hybridization with BglII-SalI digestion. In order to explaini this feature, we propose three possible configurations for pIP1510 with respect to positions of the two IS140 fragments, which are probably completely independent but could also be directly or inversely repeated. Schemes illustrating these hybridization studies are shown in Fig. 3. The data in Fig. 2 also suggest that, in the cases examined, the resistance gene clusters of the animaland human-deriyed plasmids were inserted in different regions, as indicated by the sizes of the respective PstI fragments which hybridize with the IS140 probe.

DISCUSSION
We have shown that a number of apramycinand gentamicin-resistant bacterial isolates obtained from a variety of animal and human sources from different countries are also resistant to the aminoglycoside hygromycin B. Restriction endonuclease digestion and hybridization studies show that the apramycin (aacC4) and hygromycin (hphB) genes are adjacent and clustered in the same orientation as that identified for the first apramycinand hygromycin-resistant isolate in bacteria isolated from farm animals (1). The two genes form an operon (10) and are associated with IS140, which may imply a transposable structure. IS140 belongs to the IS6 family, which was the first described family of this type (6). Such elements are generally found as a single copy of the insertion element, but in some cases a directly repeated copy is present. In the case of pIP1510 two insertion elements appear on the plasmid, of which only one could carry aacC4 linked with hphB (Fig. 3). The multiplicity of IS140 fragments on plasmids pUZ7343 and 86061324 does not permit a simple analysis of the question of transposition of the resistance gene operon.
It is probable that the strong selection pressure from the use of apramycin and hygromycin B in animals led to the emergence of this type of resistance in animals. Subsequently, the appearance in human isolates was probably due to selection by gentamicin used in both human and veteri-  plasmids) in bacteria from animals to humans has now been demonstrated in several cases (3,12). We provide plausible evidence for another example, which is of interest since two new antibiotic resistance genes in the form of an operon can be shown to be present in human clinical isolates, although previously they were found only in animals. The mechanism of this transfer is presently unknown; the apramycinand hygromycin B-resistant human and animal isolates analyzed in this study came from quite different geographical locations. While the hphB gene is of no significance in humans, since hygromycin B and its derivatives are used strictly for veterinary purposes, the aacC4 gene, which encodes resistance to a variety of aminoglycosides, including gentamicin and tobramycin, is an unwanted addition to the known armory of resistance determinants in human isolates. Clearly, the organization of resistance genes on plasmids and transposons can determine which resistance characters are transferred. In the case we describe for apramycin (or gentamicin) and hygromycin B, the association of the two genes in an operon (10) controlled by a single regulatory element guarantees their joint transfer and function. The evolution of multiple-resistance gene operons merits further investigation.