Novel β-Lactamase blaARL in Staphylococcus arlettae

Penicillins are an important group of antibiotics used to treat various types of infections caused by Gram-positive bacteria. So far, the blaZ gene was the only known β-lactamase gene in staphylococci. However, other putative β-lactamases were identified, and one of them was shown to be a novel functional β-lactamase encoded by blaARL in Staphylococcus arlettae, further limiting treatment options.

S taphylococcus arlettae is a ubiquitous coagulase-negative staphylococcus first isolated from the skin and nares of poultry and goats, respectively (1). Later, it was also found in the environment of tobacco fermentation (Culture Collection, University of Göteborg [CCUG], Göteborg, Sweden), the skin of horses (2), and bovine teat skin (3). In some cases, it was associated with bovine mastitis (4). Today, the intramammary application of penicillin alone or in combination with other antibiotics is the mastitis treatment method most frequently used in dairy cows (5). However, penicillin can be hydrolyzed by ␤-lactamase-producing staphylococci that have acquired the blaZ gene, so far the only known ␤-lactamase gene in staphylococci (6). This gene is organized in an operon with the antirepressor-encoding gene blaR1 and the repressor-encoding gene blaI. BlaR1 and BlaI form a regulatory two-component system responsible for inducible blaZ expression in the presence of ␤-lactam antibiotics (7,8). The blaZ gene is widespread in several Staphylococcus species, including Staphylococcus aureus (6,9), and has been found on different mobile genetic elements like transposon Tn552 and conjugative plasmids (10)(11)(12).
In 2010, penicillinase-producing S. arlettae strain SAN1670 was isolated from a bovine mastitis milk sample at our institute in Switzerland. PCR failed to identify the blaZ gene, prompting us to determine the nature of this ␤-lactamase phenotype by whole-genome sequencing. This allowed us to identify a novel functional ␤-lactamase in S. arlettae. Searching for further bla homologs in the gene pool of Staphylococcus revealed several uncharacterized potential ␤-lactamase sequences.
Novel ␤-lactamase bla ARL on the chromosome of S. arlettae SAN1670. The wholegenome sequence of S. arlettae SAN1670 was obtained by using Illumina MiSeq technology and reagent kit v 2 (Illumina, Inc., San Diego, CA) at the Labormedizinisches Zentrum Risch, Liebefeld-Bern, Switzerland. Reads were assembled into contigs with Geneious version R9.1.5 (13). TBLASTn analysis (http://www.ncbi.nlm.nih.gov/blast/) of the contigs generated revealed a distantly related blaZ homolog on a 145-kb contig (GenBank accession number KY363215). This blaZ homolog was named bla ARL , where bla defines the gene and ARL is the enzyme, in accordance with the nomenclature used for other ␤-lactamases (14). The 849-bp bla ARL gene encodes a 282-amino-acid protein containing the consensus pattern for the ␤-lactamase class A active site (PS00146) defined in the Prosite database (15). The active-site serine present in all class A, C, and D ␤-lactamases was identified at position 63 of the ARL enzyme. The bla ARL gene was preceded by two regulatory genes, blaI ARL and blaR1 ARL , transcribed in the opposite direction, forming a ␤-lactamase operon similar to blaI-blaR1-blaZ. This operon had 55% overall nucleotide sequence identity with Tn552 (GenBank accession number X52734) (11) and is expected to be responsible for inducible bla ARL expression in S. arlettae SAN1670. Analysis of a 50-kb region on each side of the bla ARL gene identified genes belonging to the core genome of staphylococci such as xprI, pbuX, guaA, and guaB, which are involved in purine metabolism, as well as rpsR, rpsF, and ssb, which encode ribosomal proteins and a single-strand DNA-binding protein. The absence of transposases or recombinases within this region indicates that bla ARL is stably integrated into the chromosome.
Identification of bla homologs in staphylococci. A search for ARL enzyme homology in all of the available staphylococcal sequences in the NCBI GenBank database showed that the bla ARL gene was also present in shotgun genomes of S. arlettae strains CVD059 (GenBank accession number ALWK01000016) (16) and EGD-HP3 (GenBank accession number AVOQ01000023). These bla ARL genes were 99.5% identical and had 94% nucleotide sequence identity and 97% amino acid sequence identity with bla ARL of SAN1670. Alignment of bla ARL with blaZ of S. aureus NCTC 9789 (GenBank accession number X52734) (11) resulted in only 59% nucleotide sequence identity between the genes and 48% amino acid sequence identity between the ␤-lactamases ARL and PC1 encoded by blaZ. The PC1 enzyme is widespread in staphylococci and was identified in 27 different species (Fig. 1). Additional putative ␤-lactamases containing the class A consensus pattern (PS00146) were also detected. Four of these ␤-lactamases were found in the class E mec gene complex and clustered into a group with 67 to 71% amino acid sequence identity with PC1 and 46 to 49% amino acid sequence identity with ARL (Fig. 1). The other eight uncharacterized ␤-lactamases were unrelated and had 47 to 67% amino acid sequence identity with PC1 and 47 to 56% amino acid sequence identity with ARL (Fig. 1). These putative ␤-lactamases were unique to the species they belonged to, and none of them were preceded by the regulatory genes blaI and blaR1, such as in blaZ and bla ARL operon.
Expression of bla ARL in S. aureus. To prove the functionality of the novel ␤-lactamase of S. arlettae, the bla ARL gene was cloned with and without the regulator genes blaI ARL and blaR1 ARL from SAN1670 and expressed in S. aureus RN4220. The entire blaI ARL -blaR1 ARL -bla ARL operon was amplified with primers blaR1_M1670-XhoI-F and bla_ M1670-PstI-R (see Table S1 in the supplemental material for the primers and PCR conditions used). The resulting fragment was cloned into the XhoI and PstI restriction sites of the S. aureus-Escherichia coli shuttle vector pTSSCm (17) to generate plasmid pSAN01. The bla ARL gene alone was amplified with primers bla_M1670-NdeI-F and bla_M1670-SpeI-R (see Table S1) and inserted downstream of the type 1 capsule gene 1A promoter (P cap ) of pBUS1-P cap -HC (17) to generate plasmid pSAN02. Plasmids pSAN01 and pSAN02 were transformed into E. coli DH5␣ and selected for tetracycline resistance (10 g/ml) encoded on the vectors. Sanger sequencing confirmed the correct bla ARL operon sequence in pSAN01; therefore, the plasmid was electroporated into RN4220 (18). However, nonsense mutations were observed at the 5= end of the bla ARL gene in all of the pSAN02 plasmids sequenced, indicating that constitutive ␤-lactamase expression could be deleterious to E. coli. To reverse the mutation in bla ARL from pSAN02, QuikChange site-directed mutagenesis was performed directly in S. aureus RN4220. A missing thymidine (T) in the T stretch at gene positions 10 to 15 in a faulty plasmid was introduced by PCR (Phusion Hot Start II High-Fidelity DNA Polymer-ase; Thermo Fisher Scientific, Waltham, MA) with overlapping primers mut_M1670-F (5=-GGTTTATCATATGAAAAAGTTTTTTACTATCTTTGTCTTACTCTG) and mut_M1670-R (5=-CTTTTTCATATGATAAACCTCCTATTTTCCTTTCTTGTTTTC) (the T stretch is italic, and the start codon of bla ARL is bold) (19). The reaction product was treated with the DpnI restriction enzyme and directly electroporated into RN4220 cells to obtain plasmid pSAN02mut. Sequencing of the mutagenized plasmid in RN4220 clones confirmed the correct sequence of bla ARL . Furthermore, pSAN02mut isolated from RN4220 could not be transformed into E. coli, confirming that the constitutive expression of bla ARL from P cap is not compatible with E. coli.
The MICs of both penicillin and ampicillin were higher for RN4220/pSAN02mut expressing bla ARL constitutively than for RN4220/pSAN01 containing bla ARL regulated by blaI ARL and blaR1 ARL (Table 1). Higher MICs of the cephalosporin cefoxitin and the carbapenem meropenem, with a 2-fold increase, were also observed with pSAN02mut. This is likely to be a side effect of overproduction of ARL, a protein that can bind ␤-lactams. It is unlikely that ARL can hydrolyze these ␤-lactam rings since class A ␤-lactamases like PC1 are primarily penicillinases and are not expected to have any cephalosporinase or carbapenemase activity (21). Absence of carbapenemase activity was confirmed with the Blue-Carba test (22).
Distribution of bla ARL in S. arlettae. Ten additional S. arlettae strains from different origins were tested for ␤-lactam resistance ( Table 1). All displayed decreased susceptibility to penicillin with a MIC above the CLSI resistance breakpoints (20). Production of ␤-lactamase by the nitrocefin slide method was also observed in all of the strains except SAN2420, which was negative in this test. All strains were positive for bla ARL by PCR with primers blaARL-F (5=-CTATCTTTGTCTTACTCTGTGT) and blaARL-R (5=-GCMTG ACGTGCTGCTTGTGC) (see Table S1). Analysis of the bla ARL region by PCR and Sanger  Table S1). The blaI ARL -blaR1 ARL -bla ARL operon sequences of the 10 S. arlettae strains have 88 to 100% nucleotide sequence identity with that of SAN1670.
The universal presence of bla ARL in all of the tested S. arlettae strains from different sources suggests intrinsic penicillin resistance in this species. The blaI ARL -blaR1 ARL -bla ARL operon seems to be a stable part of the core genome and not to be associated with any recombinase. However, the location between guaA and rpsR, integration hot spots for genomic islands (23,24), suggests a potential for bla ARL mobilization. In addition, diverse proteins containing typical ␤-lactamase motifs appear to be present in many different Staphylococcus species. They lack the antirepressor blaR1 and repressor blaI genes, and their role in ␤-lactam resistance is unclear. Our data propose a broader genetic analysis of penicillin-resistant staphylococci that do not contain blaZ. They also show that the presence of a functional ␤-lactamase in S. arlettae is presumable and jeopardizing penicillin treatment. The identification of the pathogen, as well as antimicrobial susceptibility testing, is therefore necessary for correct and effective therapy.
Accession number(s). The sequence of the bla ARL -containing contig of S. arlettae SAN1670 has been deposited in the GenBank database under accession number KY363215. The sequence of the blaI ARL -blaR1 ARL -bla ARL operon of S. arlettae strain ILRI338 has been deposited under accession number KY464892, and those of strains CCUG 50677, BM242, CCUG 32416, CSKR33, SAN1988, SAN2420, SAN2677, SAN2690, and CCUG 33610 have been deposited under accession numbers KY363206 to KY363214, respectively.