Resistant Gram-negatives

How should diagnostic laboratories screen for ESBL producers?

ARMRL, in consultation with other interested groups, has compiled a document entitled Guidance to Diagnostic Laboratories Laboratory Detection Reporting of Bacteria with Extended-Spectrum beta-Lactamases (PDF, 60 KB)

How should Enterobacter spp. and other AmpC producers be screened for ESBLs ?

The simplest way is to screen with cefepime and cefepime / clavulanic acid. There is an Etest strip for this combination. Cefepime is a very weak substrate for AmpC enzymes, but is a substrate for ESBLs. In the presence of clavulanate, ESBL-mediated cefepime resistance will be reduced, despite any induction of AmpC (see also the guidelines listed above).

Will Stenotrophomonas maltophilia respond to meropenem or other beta-lactams if it appears susceptible in vitro ?

No. Susceptibility tests on this species are markedly influenced by medium etc. The species produces an intrinsic class B carbapenemase that confers resistance to carbapenems. The combination of this carbapenemse and a second beta-lactamase effectively renders most beta-lactams unsuitable for this species. The treatment of choice is co-trimoxazole, although Timentin (ticarcillin/clavulanic acid) may be considered as an alternative for cotrimoxazole-resistant isolates. Note also, that the S. maltophilia commonly appears resistant to trimethoprim and sulfonamdes when they are tested separately; it usually remains susceptible to the combination.

Should imipenem-resistant Proteus spp. be referred for testing ?

Not usually. This genus is least susceptible of the Enterobacteriaceae to imipenem, with MICs hovering around the breakpoint (4 mg/L). Hence some isolates may have low-level imipenem resistance, while retaining susceptibility to meropenem and ertapenem.

Any isolates of Proteus spp. displaying resistance to meropenem or ertapenem should be submitted for confirmation and possible investigation of resistance mechanisms, as should any with high-level imipenem resistance (MIC >16 mg/L).

Who assigns numbers for novel bla alleles encoding beta-lactamases?

This is done by Karen Bush or George Jacoby. They ensure that alleles are distinct from published and novel 'in press' examples. The catalogue of designated enzymes is updated regularly, and includes e.g. phylogenetic trees of enzyme families, GenBank accession numbers for prototypes of each allele etc.

Resistant Gram-positives

Can glycopeptide-resistant enterococci be resistant to teicoplanin, but susceptible to vancomycin ?

No. There are no confirmed reports of this phenotype. GRE are always resistant to vancomycin. Cross-resistance to teicoplanin is shown by strains with VanA resistance (and very rare isolates with consitutively-expressed VanB resistance).

However, there is a potential for laboratories to over-report teicoplanin resistance in enterococci. The interpretative zone diameters recommended by the British Society for Antimicrobial Chemotherapy (BSAC) for teicoplanin against enterococci require review. Current recommendations can result in incorrect reporting of teicoplanin-resistant, vancomycin-susceptible isolates (Potz & Livermore, unpublished data).

Can staphylococci be resistant to teicoplanin, but susceptible to vancomycin ?

Yes. In fact, this is the commonest glycopeptide resistance phenotype in staphylococci, and is particularly prevalent among coagulase-negative species.

Why are some MRSA apparently susceptible to penicillin or other beta-lactams ? Can these agents be used ?

Penicillin-susceptible MRSA are usually beta-lactamase negative. Some beta-lactamase positive MRSA may appear susceptible to e.g. co-amoxiclav or imipenem.

In clinical laboratories, methicillin/oxacillin is usually tested under "inducing" conditions ( e.g. low temp, in presence of salt) to maximize expression of the mecA gene. However, penicillin and other beta-lactams are often tested on regular medium at 37oC. If these agents are tested under inducing conditions, MRSA isolates will usually appear resistant.

Clinically, MRSA and MR-CoNS should be reported resistant to all currently-available beta-lactams.

Why are some glycopeptide-resistant enterococci susceptible to ampicillin ?

Most GRE are E. faecium, but glycopeptide resistance also occurs in E. faecalis, which is ampicillin-susceptible. Intrinsically vancomycin-resistant species ( E. gallinarum, E. casseliflavus/ flavescens) are also ampicillin-susceptible.

Why can cross-resistance occur between macrolide, lincosamide and streptogramin B agents (MLSB phenotype)? What is the difference between inducible and constitutive MLSB resistance ?

This form of resistance is mediated by erm determinants that encode adenine methylases. This family of proteins methylates adenine at nucleotide 2058 in the 50S subunit of the 23S ribosomal RNA. Cross-resistance is conferred because this nucleotide is necessary for the binding of antibiotics belonging to these three structurally-unrelated chemical classes.>

Macrolides are good inducers of erm determinants, but lincosamides (like clindamycin) are not. Therefore, isolates with inducible erm genes may appear resistant to erythromycin, but susceptible to clindamycin; double disc synergy tests can be useful indicators of induction in this instance. Clear resistance to both erythromycin and clindamycin may indicate constitutive erm expression, but may be due to alternative mechanisms.

Clinically, clindamycin should not be used against erythromycin-resistant staphylococci, or streptococci as this would be expected to select for constitutive erm expression mutants. A hypothetical exception to this would be treatment of a macrolide-resistant strain that has been proven not to contain an erm gene ( not a practical consideration, and not a reference service offered by ARMRL).

Can amikacin be useful for high-level gentamicin-resistant (HLGR) enterococci ?

HLGR (MICs > =512 mg/L) is almost always mediated by a bifunctional aminoglycoside modifying enzyme, AAC(6')-APH(2"); it remains the only mechanism of HLGR reported in the UK. This enzyme confers resistance to all aminoglycosides licensed in the UK, except streptomycin. Arbekacin, which is available in Japan, is a poor substrate and retains activity against some HLGR strains.

HLGR abolishes the synergic bactericidal activity expected of aminoglycosides combined with cell wall-active agents (penicillins or glycopeptides). Screening with amikacin can lead to confusion; some HLGR isolates may not appear highly resistant in vitro even though synergy would be abolished. Thus gentamicin and streptomycin are the best aminoglycosides to test against most enterococci. Note, however, that non-HLGR E. faecalis isolates should also be screened for high-level kanamycin resistance, if amikacin is preferred over gentamicin in your hospital; HLKR again precludes synergy with amikacin combinations.

General antibiotic FAQs

Is a bacterial isolate considered susceptible to an antibiotic if its MIC is equal to the breakpoint ?

MICs less than or equal to the breakpoint concentrations are considered susceptible, MICs higher than the breakpoint indicate resistance. If two breakpoints are advocated for a particular bug/drug combination, isolates with MICs between the two values are considered to show intermediate susceptibility. Formally, therefore, answer is "Yes". However, the distribution of susceptibilities for the particular species against the drug in question should be taken into account. If, for example, the modal MIC for a given bug/drug combination is 0.06 mg/L and the breakpoint is 1 mg/L then, clearly, an isolate with an MIC of 1 mg/L shows markedly elevated resistance compared with the mode. It may harbour an acquired resistance mechanisms even though it is still formally considered to retain susceptibility.

How are MIC breakpoints set ?

See the article in the Antimicrobial Susceptibility Testing: BSAC Working Party Report by Alasdair MacGowan and Richard Wise for a discussion of criteria used by the BSAC. Other bodies will use criteria that are generally similar. EUCAST also have a document on this issue.

Is there any software specifically for analyzing susceptibility data ?

WHONET is Windows-based, and was developed for the management of microbiology laboratory data and the analysis of antimicrobial susceptibility test results. It is avaliable free.

Where can I find out about resistance genes and their nomenclature?

• aminoglycoside resistance
• beta-lactamase nomenclature and determinants
• macrolide resistance
• tetracycline resistance nomenclature and determinants