Location and organization of detected resistance genes

The location and flanking DNA sequences of resistance genes provide information about the putative origin and the transferability of this resistance gene. The distribution of identical mobile elements among different bacterial species gives information about the host range and transferability of this element.

3.2.1. erm(B)

In the four erm(B)-carrying bovine S. chromogenes isolates, the gene was located on plasmids of about 25–30 kb in size. In three cases, these plasmids also carried the lnu(A) gene. The elements on which the erm(B) gene is typically located are the closely related non-conjugative transposons Tn551 or Tn917, about 5.3 kb in size.¹¹⁸ They have been identified on medium-sized plasmids in staphylococci and streptococci, pI258 (28.2 kb)⁵³ and pAD2 (22 kb),¹⁰⁶ respectively, as well as in the chromosomal DNA.⁷⁰ Tn917 has been found in a truncated form on the smaller plasmids pAM77 (6 kb) from Streptococcus sanguis^44,106,125 and on pSES20 (8 kb) from S. lentus.¹¹⁹ Thus, the data presented in [Chapter 2] are in line with the literature.

In a study on canine and feline S. intermedius erm(B) was assumed to be exclusively chromosomally located. However, all erm(B) genes detected were shown to be associated with transposable elements other than Tn917 and Tn551.¹³

Both inducibly resistant as well as constitutively resistant isolates have been observed in that study. Inducible resistance was characterized by resistance to both erythromycin and spiramycin and reduced susceptibility to intermediate resistance to clindamycin.¹³ This was in agreement with the observations of the surveillance study presented in [Chapter 7].

S. intermedius isolates with MIC values of clindamycin between 1 and 32 mg/L were shown to express inducible clindamycin resistance in the presence of erythromycin (see subchapter 5 of the discussion).

3.2.2. erm(C)

The erm(C)-carrying plasmids detected in the study on bovine mastitis isolates [Chapter 2] belonged mainly to the group of small (2.3–2.5 kb) plasmids similar to pT48 (inducibly expressed erm(C) gene)²² and pN131 (constitutive),^55,81 on which erm(C) is usually found.⁶⁷ Those bovine CoNS isolates which carried a constitutively expressed erm(C) gene all showed a deletion of 111 bp (Figure 1, A.). This deletion comprises most of the translational attenuator – the leader peptide gene and the inverted repeats (IR) IR1–IR3. The unpaired IR4 is consequently available for a translating ribosome and thus explains the constitutive type of gene expression.

SD1 ORF of the 19 aa leader peptide SD2 erm(C)

1 2 3 4

1855 1850 1842 1783 1733 1728 1722 998

2 3 4

1862 1784 1784 1733 1728 1722 998

4

1855 1850 1845 1733 1728 1722 998

WT

A.

B.

Figure 1. Regulatory region of erm(C) of two naturally occurring plasmids in comparison with the wildtype sequence (WT) of pT48. Inverted repeats (IR1–IR4) are indicated by numbered arrows. The Shine-Dalgarno sequences of the leader peptide gene (SD1) and erm(C) (SD2) are shown as grey boxes; the reading frame for the leader peptide and erm(C) are presented as crinkled and stripped boxes, respectively. Deletions are shown as dashed lines. The numbers refer to the nucleotide positions in pT48. The last and first nucleotide still present is indicated. A. Deletion of 111 bp comprising most of the regulatory region. B. Deletion of 74 bp comprising SD1 and the leader peptide gene including IR1.

A single erm(C)-carrying plasmid differed in its size and restriction map from the latter group. The plasmid of a Staphylococcus saprophyticus isolate was slightly larger (∼3.8 kb) and the restriction map resembled that of pE194.⁴⁵ This plasmid also carried another type of deletion within the erm(C) regulatory region responsible for constitutive resistance (Figure 1, B). In this isolate the reading frame for the leader peptide including SD1 and IR1 was deleted,

excluding the last 5 bp of the leader peptide gene. In this situation, the most stable secondary structure of the mRNA is formed by IR2 and IR3; SD2 and the start codon of erm(C) located within IR4 remains accessible for the translating ribosome. Such deletions are extensively discussed in [Chapter 6].

3.2.3. msr(A) and mph(C)

Despite previous reports about msr(A)-carrying plasmids,^71,89 msr(A) was detected in the chromosomal DNA more often than on plasmids. The plasmids were about 20–30 kb in size. In each isolate that was positive for both msr(A) and mph(C), these genes were physically linked in arrangements that have been described previously.⁷² Two different types of spacer sequences were determined which resembled the corresponding regions in previously described isolates (Figure 2). The type of linkage did not correlate with the location of the genes.

msr(A) mph(C)

1343 bp

msr(A)-forw. mph(C)-rev.

SBgD B SD D VMS

msr(A) mph(C)

1093 bp

msr(A)-forw. mph(C)-rev.

S D B S Ba A

A.

B.

Figure 2. Comparison of the two detected spacer regions between the msr(A) and the mph(C) genes. The genes are shown by the bold arrows. Restriction enzymes are abbreviated as follows: A (AclI), B (BspHI), Ba (BamHI), Bg (BglII), D (DraI), M (MfeI), S (SspI) and V (VspI). The used PCR primers are indicated by the small arrows. A. The spacer sequence detected in isolates of S. xylosus, S. haemolyticus, S. simulans and S.

epidermidis, showing the best match to the corresponding sequence of pSK1 from S. aureus. B. The spacer sequence detected in two S. epidermidis isolates and one S. haemolyticus isolate, showing the best match to pM97 from S. aureus.

3.2.4. lnu(A)

The lnu(A) gene was mainly located on small plasmids. They were less than 3 kb in size (with the exception of pLNU9 with 3.8 kb). Sequence analysis revealed that they belong to the pC194 family of rolling-circle (RC) plasmids [Chapter 5]. Three S. chromogenes isolates carried lnu(A) together with erm(B) on a large plasmid. In rare cases – in a Staphylococcus warneri and in a S. epidermidis isolate – lnu(A) was detected in the chromosomal DNA.

Up to now lnu(A) has been mainly reported to be located on small plasmids,^7,83 of which a single plasmid, pBMSa1 (2.75 kb), has been sequenced completely.⁶⁸ The sequences of further lnu(A)-carrying plasmids, namely pIP855 (2.5 kb)¹⁷ and pIP856 (2.6 kb),¹⁸ have been determined only in part. The location of lnu(A) on a large plasmid, pBI109PGL (44 kb), has been described. Plasmid pBI109PGL mediated also penicillin and aminoglycoside resistance.⁷ This plasmid proved to be neither conjugative, nor mobilizable. Sequence data for pBI109PGL,⁷ however, are not available.