4. Gram Positives

4. Application of the CDS to Gram positive species

4.1. Corynebacterium species

Corynebacterium susceptibility testing is performed on Blood Sensitest Agar at 35‑37°C, in an atmosphere of 5% CO2. Slow growing isolates are incubated for 48h. Isolates resistant to benzylpenicillin 0.5 units can be tested against ampicillin 5 μg. An annular radius of < 6 mm with benzylpenicillin 0.5 units and ≥ 4 mm with ampicillin 5 μg indicates reduced susceptibility to penicillin ‑ Report as: “There is decreased susceptibility to penicillin/amoxycillin/ampicillin with the MIC between 0.25 mg/L and 2 mg/L.”

4.2. Enterococci

4.2.1. Ampicillin

Most enterococci (excluding Enterococcus faecium) are susceptible to ampicillin and will have a zone of inhibition of annular radius ≥ 4 mm and a diffuse edge around an ampicillin 5 μg disc. The corresponding MIC for these isolates is ≤ 4 mg/L (Table 11.1.a).

β-lactamase producing Enterococcus faecalis

The occurrence of rare β‑lactamase producing isolates of Enterococcus faecalis makes it essential that CDS Users assiduously adhere to the protocol for testing ampicillin 5 μg against enterococci. The reference strain, Enterococcus faecalis ATCC 29212, has a zone of inhibition of > 4 mm with a hazy edge around ampicillin 5 μg (Plate 12.1.A). β‑lactamase producing strains give a sharp edged zone of inhibition around ampicillin 5 μg (Plate 12.1.B).

NOTE: β-Lactamase producing isolates may have an inhibitory zone > 4 mm in annular radius, but will still have a sharp edge to the zone. Perform a nitrocefin‑based test to confirm the presence of β-lactamase and report the isolate resistant to ampicillin if β‑lactamase is detected.

Enterococcus faecium and ampicillin

The majority of E. faecium are resistant to ampicillin with growth up to the 5 μg disc (Plate 12.1.C). The resistance to ampicillin/benzylpenicillin in E. faecium is associated with low affinity penicillin binding proteins1. Susceptibilities are reported as per the standard interpretation.

4.2.2. Doxycycline

Doxycycline 30 μg (Oxoid, DO 30, CT0018B) has been calibrated for the use in the CDS to test organisms isolated from localised urinary tract infections2. Enterococci species are tested on Blood Sensitest agar, 35°C, 5% CO2. The MIC of susceptible strain is ≤ 16 mg/L and the annular radius of susceptible strain is ≥ 4 mm.

4.2.3. Fosfomycin

Fosfomycin/Trometamol 200 μg disc (Oxoid, FOT 200, CT0758) has been calibrated for the use in the CDS to test organisms isolated from uncomplicated UTI only (urine antiseptic). Enterococcus species are tested on Blood Sensitest agar, 35°C, 5% CO2. The MIC of susceptible strains is ≤ 64mg/L and the annular radius of susceptible strain is ≥ 6 mm.

4.2.4. Nitrofurantoin

The inclusion of a nitrofurantoin 200 μg disc when performing susceptibility testing of enterococci can assist in the differentiation of E. faecium from other enterococci when the strain under investigation is susceptible to nitrofurantoin (annular radius ≥ 4 mm)

A hazy edged zone of inhibition around a nitrofurantoin 200 μg disc with an annular radius of 5 to 7 mm indicates the identity is most likely to be E. faecium (Plate 12.1.C).

4.2.5. Trimethoprim

With the CDS Test, the testing of enterococci against trimethoprim is not recommended. In vitro, enterococci may appear to be susceptible to trimethoprim but this may not be the case in vivo. Enterococci can utilise exogenous dihydrofolate, folinic acid, tetrahydrofolate and thymidine that may be present in the urine and these compounds may antagonise the antibacterial activity of trimethoprim-sulfamethoxazole or trimethoprim. This may result not only in the failure of therapy of urinary infections caused by enterococci but also in these cases development of bacteraemia has been reported3.

4.2.6. Quinupristin/Dalfopristin

E. faecalis is inherently resistant to quinupristin and dalfopristin but the combination is usually active against E. faecium. Some isolates of E. faecium are resistant to these agents and to vancomycin.

4.2.7. Vancomycin‑Resistant Enterococci (VRE)

As a result of the emergence of “low level” vancomycin‑resistance in enterococci, important modifications have been introduced into the CDS Test for determining the susceptibility to vancomycin. “Low‑level” vancomycin‑resistant enterococci are those where greater than 90% of cells are inhibited at a concentration of 1 – 2 mg/L of vancomycin whilst the remaining 5‑10% is inhibited at a concentration of 8 mg/L. As a result, there is a marked inoculum effect i.e., the higher the inoculum, the higher the MIC. For these reasons, a low inoculum may lead to an error when determining the susceptibility to vancomycin. Therefore, it is essential that the correct CDS inoculum be used (107 cfu/mL – ensure cellular material is visible on the tip of the wire or prepare a 1 in 5 dilution of a suspension equivalent to McFarland standard 0.5).

It is mandatory that the strain under investigation be compared with the reference strain (Enterococcus faecalis ATCC 29212 which has a sharp edged inhibitory zone of > 2 mm in annular radius around the vancomycin 5 μg disc). The interpretation of the susceptibility is based on the characteristics of the inhibitory zone edge as well as the size of the zone.

The following patterns are seen when testing vancomycin 5 μg against enterococci.

  • Susceptible to vancomycin:
    1. E. faecalis generally have a zone > 2 mm with a sharp edge similar to that of the Enterococcus faecalis ATCC 29212 (Plate 12.1.A)
    2. E. faecium have a vancomycin zone up to 4 to 6 mm with a sharp edge (Plate 12.1.C).
  • Resistant to vancomycin of vanA phenotype:

These strains grow up to the edge of the vancomycin disc and the organism is also resistant to teicoplanin although some strains may have a zone of less than 2 mm around the teicoplanin 15 μg disc but strains with heterogeneous resistance may have a zone up to 6 mm with a very fine growth advancing towards the disc (Plate 12.2.B).

  • Resistant to vancomycin of vanB phenotype:
    1. E. faecalis of vanB phenotype usually have a reduced zone of less than 2 mm with a hazy edge (growth at the edge of the inhibitory zone) and therefore should be easily recognised.
    2. E. faecium of vanB phenotype may have an inhibitory zone of up to 3 mm in annular radius with a light growth advancing near vancomycin 5 µg disc when measured from the edge of confluent growth (Plate 12.2.A). Note that incubation in 5% CO2 enhances the fine growth thus the resistance will be more obvious at 24 hours than the incubation in air. In all cases, if there is doubt on interpreting the result at 24 hours, the plate is incubated for a further 24 hours to observe the light growth advancing towards vancomycin 5 µg disc.
  • Resistant to vancomycin of vanC phenotype

The organism has a sharp edged zone considerably smaller than that of the control strain. This is typical of Enterococcus gallinarum and Enterococcus casseliflavus possessing the natural vanC type resistance (Plate 12.2.C). These strains are considered resistant to vancomycin. E. gallinarum, E. casseliflavus and E. faecium do not utilise pyruvate; E. faecalis does.

NOTE: The term VRE refers only to E. faecalis and E. faecium that have acquired resistance to vancomycin. Leuconostoc and Pediococcus species are inherently resistant to vancomycin (Plate 12.2.D). VRE can be distinguished from Leuconostoc and Pediococcus by its pyrrolidonyl arylamidase (PYR) activity (See section 4.4).

4.2.8.   High level aminoglycoside resistance in E. faecalis:

All enterococci are known to be intrinsically resistant to aminoglycosides and all isolates would have a zone < 6 mm with a gentamicin 10 ug disc (CN 10). Therefore, enterococci are not calibrated against CN 10.  However, gentamicin (CN 200) and streptomycin (S 300) discs have been calibrated in the CDS to detect high level resistance to these aminoglycosides in isolates of E. faecalis from blood cultures in patients with suspected endocarditis. If the isolate does not have high level resistance, gentamicin or streptomycin may be used to provide synergy to ampicillin.  Note that the high level resistance is mediated by a different mechanism in each of the two aminoglycosides, therefore if required both should be tested.

4.3. Erysipelothrix species

Erysipelothrix species are included in the Table of Calibrations with streptococci (Table 11.1.a).

4.4. Leuconostoc and Pediococcus

Leuconostoc and Pediococcus species have high inherent resistance to both vancomycin and teicoplanin, i.e., there is no zone of inhibition observed around either a vancomycin 5 μg or a teicoplanin 15 μg disc (Plate 12.2.D). Leuconostoc and Pediococcus species lack pyrrolidonyl arylamidase (PYR) activity and this can be used to differentiate them from VRE and other enterococci.

4.5. Staphylococci

Most staphylococci grow well on Sensitest agar. Two notable exceptions include Staphylococcus lugdunensis and Staphylococcus pseudintermedius. In such cases, susceptibility testing should be performed on blood Sensitest. Reference ranges are provided in Table 11.3.a.

4.5.1. Cefoxitin & oxacillin (methicillin)

Methicillin-resistant (mecA or mecC gene positive) Staphylococcus aureus and Staphylococcus lugdunensis can be identified using cefoxitin 10 μg discs. Coagulase-negative staphylococci (excluding Staphylococcus lugdunensis and Staphylococcus saprophyticus) are tested against oxacillin 1 μg discs.

When the identification is not available at the time of susceptibility testing both cefoxitin 10 μg and oxacillin 1 μg should be used and the appropriate result reported once the identification is known.

4.5.2. Ceftaroline

Ceftaroline 5 μg disc (Oxoid, CPT 5, CT1942B) has been calibrated for the use in the CDS to test MRSA. S. aureus are tested on Sensitest Agar in air at 35-37°C for 24 hours. The MIC of susceptible strain is ≤ 1mg/L and the annular radius of susceptible strain is ≥ 6 mm.

4.5.3. Borderline oxacillin resistant Staphylococcus aureus (BORSA)

Some mecA gene negative isolates of S. aureus may produce large amounts of β-lactamase, which make them appear resistant to oxacillin because this agent is less resistant to hydrolysis by staphylococcal β-lactamase than many other β-lactams. These strains are termed borderline oxacillin-resistant S. aureus (BORSA). Cefoxitin is not affected by staphylococcal β-lactamase to the same extent and the use of a cefoxitin 10 μg disc in the CDS test allows a better differentiation of mecA gene negative from mecA gene positive S. aureus.

4.5.4. Staphylococcus aureus with low β-lactamase activity

The annular radius of the inhibitory zone around benzylpenicillin 0.5 units (P 0.5u) with β-lactamase negative S. aureus, such as S. aureus NCTC 6571, is about 12 mm (Plate 12.3.A) and the edge of the inhibitory zone is hazy.

There are rare strains of S. aureus that produce low levels of β-lactamase and for these strains, the annular radius of the zone of inhibition around benzylpenicillin 0.5 units (P 0.5u) may be as large as 4 to 5 mm zones of inhibition, but still will have a sharp edge (Plate 12.3.B). If the inoculum is too light it may result in semi-confluent lawn of growth with an inhibitory zone annular radius of up to 7 mm. However, the edge of the inhibitory zone is still sharp and these strains must be reported as resistant to penicillin and the test should be repeated. If there is any doubt, perform a β-lactamase detection test (eg. Nitrocefin test).

4.5.5. Methicillin susceptible (mecA gene negative) Staphylococcus aureus (MSSA)

Methicillin susceptible (mecA gene negative) strains of S. aureus have a zone of inhibition around cefoxitin 10 μg with an annular radius of > 6mm (usually between 8 and 9 mm). These isolates are reported as susceptible to methicillin (Plate 12.3.C).

Some strains of S. aureus are mecA gene negative and yet show multiple resistance to other antibiotics. These strains are often loosely referred to as ‘Ex-Methicillin-Resistant S. aureus’.

The annular radius of the zone of inhibition around a cefoxitin 10 μg disc is clearly > 6 mm (Plate 12.3.D). They are reported as susceptible to methicillin.

4.5.6. Methicillin resistant Staphylococcus aureus (MRSA)

Although methicillin discs are no longer available, S. aureus strains possessing the mecA gene are still referred to as methicillin resistant S.aureus or MRSA. The annular radius of the inhibitory zone around a cefoxitin 10 μg disc is  < 6 mm (usually 0 to 3 mm) for these organisms. (Plate 12.4.A, Plate 12.4.B and Plate 12.4.C). With the use of cefoxitin 10 μg discs, neither Mannitol Salt Agar nor incubation at 30°C is required to detect methicillin resistance. Report these isolates as resistant to methicillin and all other β-lactams.

Multi‑resistant MRSA

Multi‑resistant MRSA strains are often isolated from institutionalised patients and are resistant to a large number of antibiotics, such as erythromycin, tetracycline, ciprofloxacin, trimethoprim-sulfamethoxazole and gentamicin.

Non multi‑resistant MRSA

MRSA strains that are resistant only to benzylpenicillin and methicillin i.e., non-multi resistant MRSA (NMMRSA) or Community Acquired MRSA (CA-MRSA) are being isolated with increasing frequency from patients in the community and may be implicated in hospital acquired infections. Typically, these strains have no zone of inhibition or a reduced zone of between 2 and 4 mm in annular radius around a cefoxitin 10 μg disc (Plate 12.4.B). These strains may also be resistant to erythromycin, tetracycline and/or ciprofloxacin.

4.5.7. Vancomycin resistance in Staphylococcus aureus

Vancomycin resistant Staphylococcus aureus (VRSA)

VRSA was first isolated in the USA in 20024.  The isolate was resistant to oxacillin/methicillin and vancomycin (MIC > 128 mg/L). A second VRSA strain was isolated in New York in 2004. These strains will exhibit either no zone or a zone of light growth right up to the edge of a vancomycin 5 μg disc.

Vancomycin intermediate Staphylococcus aureus (VISA/GISA)

MRSA with reduced susceptibility to vancomycin and teicoplanin, known as VISA or GISA (vancomycin or glycopeptide intermediate S. aureus) have been described overseas5.  These strains do not have the same mechanism of resistance to glycopeptides that occurs in Enterococcus faecalis and Enterococcus faecium. Electron microscopy has revealed a thickened cell wall that traps glycopeptide molecules thereby blocking access to the target site. The MIC of vancomycin determined by agar dilution for such strains was between 4 and 8°mg/L.

Vancomycin susceptible staphylococci have a sharp edged zone of inhibition of > 2 mm in annular radius around vancomycin 5 μg and teicoplanin 15 μg discs. VISA strains produce a hazy edged zone of inhibition of < 2 mm in annular radius around vancomycin and teicoplanin discs. i.e., there is fine growth at the edge of the zones of inhibition. This effect is more apparent with teicoplanin than vancomycin. If in doubt, incubate the plates for a further 24 hours (Plate°12.4.C).

h- VISA

A small percentage of cells in the bacterial population of some MRSA isolates have reduced susceptibility to vancomycin but the MIC for the population, as determined by the standard agar dilution technique, remains low (2 mg/L)6. These strains are referred to as hetero or h-VISA.  Detection of this sub-population with reduced susceptibility can be difficult. The h-VISA strains will not be readily detected during routine laboratory susceptibility testing using the CDS test after 24 or 48 hours of incubation. Exposed to a vancomycin concentration gradient, cells with reduced susceptibility may multiply and become visible after 48 to 72 hours of incubation. It is important to be aware that if treatment with a glycopeptide has failed, h-VISA may be present. In this situation, isolates should be referred to a specialised laboratory.

4.5.8. Erythromycin, clindamycin and Staphylococcus aureus

S. aureus resistant to erythromycin are primarily (98%) of the MLSB phenotype (either constitutive or inducible) and are therefore also resistant to clindamycin and lincomycin (Plate 12.4.D). The remaining 2% possess an efflux mechanism that does not confer resistance to clindamycin. See section 4.7.

4.5.9. Coagulase negative staphylococci (CoNS)

In determining methicillin susceptibility for coagulase-negative staphylococci (excluding S. lugdunensis and S. saprophyticus) consider only the zone around the oxacillin 1 μg disc; ignore the zone around the cefoxitin 10 μg disc. Note: S. lugdunensis and S. saprophyticus are excluded from the following discussion.

Methicillin susceptible CoNS

Methicillin susceptible (mecA gene negative) coagulase negative staphylococci (excluding S. lugdunensis and S. saprophyticus) have a zone of inhibition around oxacillin 1 μg with an annular radius of > 6 mm (usually between 7 and 10 mm). These isolates are reported as susceptible to methicillin.

Methicillin resistant CoNS

The majority of methicillin resistant (mecA gene positive) coagulase negative staphylococci (excluding S. lugdunensis and S. saprophyticus) have a zone of inhibition around oxacillin 1 μg with an annular radius of < 6 mm (usually between 0 and 4 mm). These isolates are reported as resistant to methicillin.

4.5.10. Rifampicin and sodium fusidate/fusidic acid

Rifampicin and sodium fusidate/fusidic acid are frequently used in combination to treat infections caused by methicillin resistant S. aureus and coagulase negative staphylococci. The mutation rate of resistance for each antibiotic is high, in the order of 10-5 to 10-7 and colonies will often be observed within the inhibitory zones around both rifampicin 1 μg and fusidic acid 2.5 μg. If the zones of inhibition around rifampicin and fusidic acid are ≥ 6 mm report the isolate susceptible to the individual antibiotics.

It is advisable that a warning such as “Rifampicin and sodium fusidate must be given in combination since resistance will develop rapidly to either agent if used alone” be issued when reporting the susceptibility of these two antibiotics.

4.5.11. Staphylococcus saprophyticus from urine

It is recommended that a novobiocin 5 μg disc be included for testing staphylococci isolated from urine specimens. Urine isolates of coagulase negative staphylococci resistant to novobiocin (annular radius < 4 mm) may be presumptively identified as S. saprophyticus. S. saprophyticus is a special case where benzylpenicillin 0.5 units and oxacillin 1 μg discs are not used for testing. The MICs of benzylpenicillin and oxacillin are relatively high with wild strains of S. saprophyticus isolated from urine when compared with other staphylococci, i.e. they are intrinsically less susceptible to all penicillins and cephalosporins. Also, some isolates produce very low levels of a non‑inducible penicillinase. For these reasons, the annular radius of the inhibitory zone around benzylpenicillin 0.5 units and oxacillin 1 μg discs recorded with susceptible strains of S. saprophyticus may be < 6 mm and therefore these two discs are not used for testing this species.

Ampicillin 5 μg (instead of benzylpenicillin 0.5 u) and cephalexin 100 μg (instead of oxacillin 1 μg) discs are therefore used for the testing of this species and cephalexin is used as the surrogate disc for reporting the susceptibility to amoxycillin-clavulanic acid.

S. saprophyticus may possess a non‑inducible or an inducible β-lactamase or no β-lactamase at all. In addition, the mecA gene may or may not be present.

Results that may be obtained from testing ampicillin and cephalexin against S. saprophyticus are:

  • Susceptible to both ampicillin and cephalexin.

The isolate does not have the mecA gene and possesses either no β-lactamase or a non‑inducible β-lactamase (Plate 12.5.A).

  • Resistant to ampicillin but susceptible to cephalexin.

The isolate does not have the mecA gene but does possess an inducible β-lactamase (Plate 12.5.B).

  • Resistant to ampicillin and cephalexin.

The isolate has the mecA gene (Plate 12.5.C).

4.6. Streptococci

4.6.1. Streptococcus pneumoniae

Five enzymes in the cell wall of S. pneumoniae, the penicillin‑binding proteins (PBP 1A, 1B, 2A, 2B and 2X) are the target sites for β-lactam antibiotics. Increases in the MIC of benzylpenicillin and cefotaxime/ceftriaxone are the result of changes in one or more of the PBPs. Although S. pneumoniae strains resistant to cefotaxime/ceftriaxone are often resistant to benzylpenicillin, the correlation is not perfect. A similar situation applies with oxacillin and benzylpenicillin. For these reasons, both benzylpenicillin (not oxacillin) and cefotaxime/ceftriaxone should be tested. Testing and interpretation of susceptibility results are dependent on the site of isolation.

CSF

  • Benzylpenicillin: Isolates are tested using a benzylpenicillin 0.5 u disc (P 0.5u). Only isolates with an annular radius of the zone of inhibition ≥ 6 mm are reported susceptible to benzylpenicillin. The MIC of benzylpenicillin of susceptible strains is ≤ 0.125 mg/L.
  • Cefotaxime or ceftriaxone: Isolates are tested using a cefotaxime or a ceftriaxone 0.5 μg disc. Only isolates with an annular radius of the zone of inhibition ≥ 4 mm are reported susceptible to cefotaxime or ceftriaxone. The MIC is ≤ 0.5 mg/L.

Sites other than CSF

Isolates from sites other than CSF (sputum, ear, eye and blood cultures not associated with meningitis) are tested against an ampicillin 5 μg disc and a higher potency cefotaxime or ceftriaxone 5 μg disc (Table 11.1.a) in addition to those used for CSF isolates.

  • Benzylpenicillin 0.5 u/ampicillin 5 μg: If the inhibitory zone is < 6 mm with a benzylpenicillin 0.5 u disc and ≥ 4 mm with an ampicillin 5 μg disc, report the susceptibility as follows: “There is decreased susceptibility to penicillin, ampicillin and amoxycillin with the MIC between 0.25 mg/L and 2.0 mg/L” (Table 1.a).
  • Cefotaxime or ceftriaxone 0.5 μg /cefotaxime or ceftriaxone 5 μg: if the inhibitory zone is < 4 mm with a cefotaxime or a ceftriaxone 0.5 μg disc and ≥ 6 mm with a cefotaxime or a ceftriaxone 5 μg disc, report the susceptibility as follows: “There is decreased susceptibility to cefotaxime (or ceftriaxone) with the MIC between 1.0 mg/L and 2.0 mg/L”.
  • Note: The “susceptible” breakpoints of ≤ 0.125 mg/L for benzylpenicillin and ≤ 0.5 mg/L for cefotaxime/ceftriaxone were established with wild type isolates of S. pneumoniae and correlated well with clinical response. Strains with a diminished susceptibility to these agents appeared after the initial calibrations and there is a dearth of strong clinical evidence to indicate that infections with these strains would respond to treatment with the antibiotics concerned. This should be kept in mind when interpreting susceptibility testing reports of “decreased susceptibility” for S. pneumoniae.

4.6.2. Other streptococci

The susceptibility of β‑haemolytic streptococci of groups A, C, G to the penicillins and cephalosporins (except ceftazidime as Gram‑positive organisms are resistant to this cephalosporin) is extrapolated from benzylpenicillin 0.5 u (Table 12.2.a). Other streptococci including group B streptococci, S. mitis, S. sanguinis and S. anginosus (formerly S. milleri) group showing resistance to benzylpenicillin 0.5 u or CTX 0.5 μg can be tested against ampicillin, cefotaxime and ceftriaxone 5 μg discs and interpreted as for S. pneumoniae (Section 4.6.1).

NOTE: If infective endocarditis is present, the MIC should be determined.

4.7. Erythromycin and clindamycin

Three mechanisms have been found amongst Gram positive bacteria that confer resistance to the macrolides (erythromycin, roxithromycin, clarithromycin, and azithromycin) and the lincosamides (clindamycin, lincomycin) 7, 8.

  • Methylation of the 23S ribosomal subunit (the target site of action of macrolides and clindamycin) confers resistance to all macrolides and clindamycin. This is the MLSB phenotype (M = macrolide, L = lincosamide, SB = streptogramin B). Resistance may be constitutive (cMLSB phenotype) or inducible (iMLSB phenotype).
  • An efflux pump promotes the efflux of macrolides but not clindamycin from the cell. This is the M phenotype.
  • The third mechanism of resistance is unknown. It confers resistance to clindamycin but not to erythromycin. This is the LSA (L=lincosamide, SA= streptogramin A) phenotype described for Streptococcus agalactiae9.

When erythromycin and clindamycin are tested against Gram‑positive organisms one of five susceptibility profiles is seen.

  1. The isolate is susceptible to both erythromycin and clindamycin.The isolate is reported as susceptible to clindamycin, erythromycin and all other macrolidesWhen S. aureus was exposed to erythromycin, resistant mutants arose at a high frequency of 10-5 to 10-6. In contrast, no clindamycin resistant mutants arose when 109 cfu of S. aureus were exposed to clindamycin. It is likely that clinical infections with organisms of this phenotype will respond to clindamycin.

    NOTE: We recommend that in this case the susceptibility of staphylococci to erythromycin, roxithromycin or clarithromycin is not reported, instead report clindamycin. If a Gram‑positive organism is susceptible to erythromycin then, in all probability, it will be susceptible to clindamycin (except on rare occasions where the LSA phenotype might be present ‑ S. agalactiae).

  2. The isolate shows resistance to both erythromycin and clindamycin.The isolate is reported resistant to all macrolides and clindamycin (cMLSB phenotype).
  3. The isolate shows resistance to erythromycin but appears susceptible to clindamycin. Adjacent disc testing with clindamycin 2 μg and erythromycin 5 μg positioned 13 mm apart (edge to edge) shows a flattening of the clindamycin inhibitory zone adjacent to the erythromycin disc (Plate 12.4.D)The isolate has inducible clindamycin resistance (ICR positive) and is reported as resistant to erythromycin and clindamycin. ICR positive streptococci, corynebacteria, anaerobes and staphylococci can be detected in this way.Exposing aureus with this inducible iMLSB phenotype to clindamycin gave rise to a high frequency of resistant mutants (10-5 to 10-6). The MIC of clindamycin for these mutants was 16 mg/L.
  4. The isolate shows resistance to erythromycin but appears susceptible to clindamycin with no flattening of the inhibitory zone adjacent to an erythromycin disc.The isolate is of the M phenotype and is ICR negative. There is efflux of erythromycin and other macrolides but not clindamycin from the cell. The isolate is reported as resistant to erythromycin and all other macrolides but susceptible to clindamycin.Clindamycin therapy may be successful under certain clinical circumstances.

    The M phenotype is uncommon in aureus and MRSA, occurring in only 1 to 2% of erythromycin resistant strains, the remainder being of the cMLSB or iMLSB phenotypes and therefore resistant to all macrolides and clindamycin. Consequently, there is no need to routinely test for the M phenotype (by adjacent disc testing as described above) with S. aureus and MRSA isolates. However, adjacent disc testing can be performed where the mechanism of resistance is of interest (cMLSB, iMLSB or M phenotype).

  5. The isolate appears susceptible to erythromycin but resistant to clindamycin.This is the rare LSA phenotype, described in agalactiae. The mechanism of resistance is unknown9.Further information on the phenotypes of clindamycin susceptibility can be found in Newsletter°

References

1       Williamson, R., Le Bouquenec, C., Gutmann, L. & Horaud, T. 1985. One or two low affinity Penicillin‑binding‑proteins may be responsible for the range of susceptibility of Enterococcus faecium to benzylpenicillin. J. Gen. Microbiol. 131, 1933‑40.

  1. Bell, S., Pham, J., Saab, J., Nguyen, T. 2012. Calibration of doxycycline for use in urinary tract infections with enterococci. Pathology. 44(7), 654-675.

3       Murray, B.E. 1990. The life and times of the Enterococcus. Clinical Microbiology Reviews. 3(1), 46‑65.

4       Centers for Disease Control and Prevention. 2002. Staphylococcus aureus resistant to vancomycin ‑ United States, 2002. Morbidity and Mortality Weekly Report. 51(26), 565‑67.

5       Hiramatsu, K., Hanaki, H., Ino, T., Yabuta, K., Oguri, T. & Tenover, FC. 1997. Methicillin‑resistant Staphylococcus aureus clinical strain with vancomycin reduced susceptibility. J. Antimicrob Chemother. 40(1), 135‑36.

6       Ward, P.B., Johnson, P.D., Grabsch, E.A., Mayall, B.C. & Grayson, M.L. 2001. Treatment failure due to methicillin‑resistant Staphylococcus aureus (MRSA) with reduced susceptibility to vancomycin, Med. J. Aust. 175(9), 480‑83.

7       Hamilton-Miller, J.M.T. & Shah, S. 2000. Patterns of phenotypic resistance to the macrolides‑lincosamide‑ketolide‑streptogramin group of antibiotics in staphylococci. J. Antimicrob. Chemother. 46(6), 941‑49.

8       Tait-Kamradt, A., Davies, T., Appelbaum, P.C., Depardieu, F., Courvalin, P. et al. 2000. Two new mechanisms of macrolides resistance in clinical strains of Streptococcus pneumoniae from Eastern Europe and North America. Antimicrob. Agents. Chemother. 44(12), 3395‑401.

9       Malbruny, B., Werno, A.M., Anderson, T.P., Murdoch, D.R. & Leclercq. R. 2004. A new phenotype of resistance to lincosamide and streptogramin A‑type antibiotics in Streptococcus agalactiae in New Zealand. J. Antimicrob. Chemother. 54, 1040‑44.