5. Gram Negatives

Application of the CDS to Gram negative species

5.1. Acinetobacter species

Acinetobacter species can be separated into two groups on the basis of their susceptibility to ampicillin and cephalexin.

  • The ampicillin and cephalexin susceptible lwoffi and A. lwoffi like group.

This group has an inhibition zone of approximately 8 mm in annular radius around ampicillin 25 μg discs, and smaller zones around cephalexin 100 μg (7 – 8 mm) and cefotaxime 5 μg (6mm). They are resistant to trimethoprim. See Plate 12.6.A.

  • The ampicillin and cephalexin resistant baumannii and A. baumannii like group.

This group possesses a non‑inducible cephalosporinase of the AmpC type. Derepressed β-lactamase producing mutants are not found. They are resistant to ampicillin and cephalosporins. Urinary isolates may be recognised by their susceptibility to amoxycillin-clavulanic acid and resistance to cephalexin and trimethoprim. See Plate 12.6.B.

There have been reports of multi‑resistant strains of A. baumannii or A. baumannii like organisms possessing plasmid mediated zinc‑metallo‑carbapenemases (IMP‑4, inhibited by EDTA but not by clavulanic acid) or (OXA types of β-lactamase not inhibited by EDTA and variably inhibited by clavulanic acid)1,2.  Functionally similar enzymes have been found in the Enterobacteriaceae, P. aeruginosa and other organisms (See Section 5.5.8).

Both groups (even the β‑lactamase negative A. lwoffi like group) have some degree of resistance to cephalosporins. The cephalosporinase produced by Acinetobacter species is not inhibited by clavulanic acid. amoxycillin-clavulanic acid and ticarcillin-clavulanic acid belong to the β-lactam/ β-lactam inhibitor combination group of antimicrobials.  These combination groups contain a β-lactam and a second agent that has minimal activity of its own but potentiates the activity of the β-lactam to act as an inhibitor of some β‑lactamases.  The MIC of clavulanic acid ranges from 1 to 2 mg/L with the A. lwoffi / A. lwoffi like group and from 4 to 32 mg/L with the A. baumannii / A. baumannii like group.  Susceptibilities are reported according to the standard 6 mm annular radius interpretation.

Some Acinetobacter species (NOT A. lwoffi or A. lwoffi-like which are β-lactamase negative) produce a low level cephalosporinase of AmpC type. These organisms are resistant to cephalexin 100 µg (inhibitory zone annular radius 2- 3 mm) but may appear susceptible to ampicillin 25 µg with a zone marginally > 6 mm. They should be reported as resistant to ampicillin.

Note: We recommend the testing in parallel of ampicillin 25 µg and cephalexin 100 µg discs with all Acinetobacter species. See Power points ASM 2011 (on the CDS website).

5.2. Aeromonas species

Two chromosomal β-lactamases A1 and A2 have been described in Aeromonas species.

A2, a carbapenemase, hydrolyses carbapenems but not cephalosporins or cephamycins. It may show heterogeneous expression of resistance and consequently, resistance to imipenem and meropenem may not be detectable by any conventional method, including MIC determination, and false reporting of susceptibility to carbapenems may occur. (See Plate 12.7.A and Plate 12.7.B). A. caviae strains do not possess the A2 carbapenemase and can be tested against carbapenems3. All other Aeromonas species should be reported as resistant to carbapenems.

A1, an inducible cephalosporinase inhibited by aztreonam but not by clavulanic acid, hydrolyses cephalosporins and cephamycins but not carbapenems (AmpC/Bush group 1) and can be detected by adjacent disc testing (Plate 12.7.A). A close examination revealed that members of Aeromonas sp, (except A. sobria/veronii both of which failed to produce the cephalosporinase A1), yielded hyper-producing of AmpC mutants at an extremely variable rate when exposed to cefotaxime, ticarcillin-clavulanic acid, piperacillin-tazobactam, cefpodoxime, cefotetan, cefoxitin, cefuroxime or cephalexin.  Within each species the rate varied from 105 to 108.  On the other hand, we were unsuccessful in our attempts to obtain any hyper-producing mutants from the same strains by exposure to ceftazidime or aztreonam. Also, when the hyper-producing mutants that were selected by any of the β-lactams listed above were tested against ceftazidime or aztreonam, all were susceptible to these two antibiotics. It appears that the substrate specificity of AmpC of Aeromonas sp. is different to that of the AmpC of the EEC group (see section 5.5.7). Based on these observations we recommend that it is only safe to report ceftazidime, aztreonam susceptibility as well that of the 4th generation cephalosporins and carbapenems, the latter only in the case of A. caviae. (See Table 11.4.a guide to the reporting of β-lactam antibiotics).

5.3. Burkholderia pseudomallei

Isolates of this species possess an inducible chromosomal cephalosporinase that is inhibited by clavulanic acid. They are usually susceptible to amoxycillin-clavulanic acid and ceftazidime. These antibiotics together with trimethoprim-sulfamethoxazole are used for treatment. Test      B. pseudomallei using the criteria applied to Pseudomonas species with the exception of amoxycillin-clavulanic acid 60 μg (Table 11.1.b) and report as recommended in Table 11.4.a.

5.4. Chryseobacterium species (formerly Flavobacterium species)

C. meningosepticum (formerly Flavobacterium meningosepticum) and C. indologenes (formerly Flavobacterium indologenes) are the most commonly encountered species of this genus. These species are often resistant to β-lactams and may possess a zinc‑metallo‑carbapenemase. Both species are also resistant to aminoglycosides (Plate 12.9.B) and can be resistant to quinolones4.

Trimethoprim-sulfamethoxazole is the antibiotic of choice for the treatment of infections with Chryseobacterium species. Isolates typically show a large zone of inhibition around the trimethoprim-sulfamethoxazole 25 μg discs (Plate 12.9.B). Chryseobacterium species are tested using the criteria applied to Pseudomonas species (Table 11.1.b) and reported using the recommendations in Table 11.4.a.

5.5. Enterobacteriaceae

An important mechanism of resistance to β-lactam antibiotics in Enterobacteriaceae and Vibrionaceae is the production of β-lactamases. The number of identified significant β-lactamases has increased considerably with a consequential increase in the complexity of their classification. The use of a heavier inoculum and the careful selection of appropriate disc potencies in the CDS method facilitate the in vitro demonstration of β-lactamase mediated resistance. Adjacent disc testing and inhibitory zone morphology, both integral parts of the CDS method, assist in the identification of these β-lactamases.

Testing in parallel of ampicillin 25 µg and cephalexin 100 µg discs is strongly recommended with all members of Enterobacteriaceae. Strains that are resistant to cephalexin but appear susceptible to ampicillin (presence of chromosomal inducible AmpC β-lactamase) are reported as resistant to ampicillin/amoxycillin. (See Power point ASM 2011 on website).

A note about meropenem and imipenem: Meropenem has replaced imipenem as a therapeutic agent in Australia so laboratories should report the susceptibilities to meropenem and not Imipenem. The CDS recommendation is to continue to test with both meropenem 5 µg and Imipenem 10 µg but not to use Imipenem as a surrogate for meropenem because some strains of Proteus spp., Providencia spp. and Morganella morganii that are resistant to Imipenem are susceptible to meropenem.  The imipenem 10 µg disc is useful in the assessment of zone morphology (an essential part of the CDS), for example, inducible cephalosporins are most readily detected by the placement of an Imipenem 10µg disc adjacent to the cefotaxime 5 µg disc.

5.5.1. Penicillinase (broad spectrum) β‑lactamases

TEM‑1, TEM‑2 (E. coli, P. mirabilis, C. koseri) and SHV‑1 (K. pneumoniae) are penicillinases of Ambler class A (Bush group 2b) commonly found in the specified organisms. They are inhibited by clavulanic acid and confer resistance to ampicillin and cephalothin but not to cephalexin or the extended spectrum cephalosporins (Plate 12.8.A).

5.5.2. Inhibitor‑resistant TEM β‑lactamases (IRTs)

Resistance to amoxycillin-clavulanic acid in some isolates of Escherichia coli and rare isolates of Klebsiella pneumoniae might be due to the production of mutant forms of TEM‑1 β-lactamase. These TEM β-lactamases are far less susceptible to inhibition by clavulanic acid than the original TEM enzyme and are called inhibitor‑resistant TEM β-lactamases or IRTs. IRT producing E. coli are resistant to amoxycillin-clavulanic acid, ticarcillin-clavulanic acid, piperacillin-tazobactam but remain susceptible to cephalexin (Plate 12.8.B).

5.5.1. Plasmid mediated AmpC

Non‑inducible plasmid mediated cephalosporinases of Ambler class C or AmpC (Bush group 1) with varying activity have been found in Escherichia coli, Klebsiella pneumoniae and Salmonella species (Plate 12.8.C, Plate 12.8.D)5.  These enzymes are inhibited by aztreonam and boronic acid but not by clavulanic acid.

Screening for AmpCs

Plasmid mediated AmpC producers such as E.coli, and K.pneumoniae can be recognised in routine testing. They are susceptible to cefepime (a 4th generation cephalosporin), and resistant to amoxycillin-clavulanic acid 60 µg and cephalexin 100 µg. Those with high AmpC activity are also resistant to cefotaxime 5 µg and ceftazidime 10 µg.

The susceptibilities are reported according to the standard interpretation.

Confirmation of AmpC (optional)

The use of boronic acids has long been recognised as a specific inhibitor of AmpC β-lactamases6,7.  The expression of an AmpC can be confirmed by demonstrating synergy between a disc containing 200 ug of boronic acid and an adjacent cefotaxime 5 µg, amoxycillin-clavulanic acid 60 µg, cephalexin 100 µg and ceftazidime 10 µg disc (Plate 12.9.C, Plate 12.9.D; see Power points ASM 2010 on the CDS website).

Note: To prepare a solution containing 200 µg in each disc (i.e. 8 mg/ml), weigh 400 mg boronic acid (Thianaphthene-2-boronic acid from Sigma – Product number: 499978) and dissolve the powder in 50 ml 70% ethanol in a volumetric flask. Sterilise by filtering through a membrane filter 0.45 µm. Dispense in 10 ml aliquots. The solution is stable in the dark at room temperature.

Boronic acid discs used in phenotypic detection of AmpC are prepared by lining up 6mm blank paper discs in a Petri dish. Drop 25 µL boronic acid onto each disc. Let dry for 24-48 hours (until dry) with lid on. Keep Petri dish protected from light. Transfer dry discs to a jar and store in a drawer.  This solution cannot be applied to antibiotic discs because of the presence of ethanol.

5.5.2. Extended spectrum β‑lactamases (ESBLs)

The abbreviation ‘ESBL’ is used to refer to plasmid mediated extended spectrum β-lactamases, though there are reports of these integrating into the bacterial chromosome. These enzymes can hydrolyse extended spectrum cephalosporins and aztreonam but do not hydrolyse the cephamycins (cefoxitin and cefotetan). They are inhibited by clavulanic acid and can be acquired by all members of the Enterobacteriaceae. ESBL production can be detected by adjacent disc testing: position a disc containing clavulanic acid (amoxycillin-clavulanic acid 60 μg or ticarcillin-clavulanic acid 85 μg) adjacent to a cephalosporin disc. An elliptical clearing between the two discs indicates the inhibition of the β-lactamase by clavulanic acid (Plate 12.10.A and Plate 12.10.B). This synergistic effect may be subtle and harder to detect in isolates with a high activity ESBL (no zone around a CTX 5). In these cases, repositioning the discs slightly closer together can intensify the effect and confirm the presence of an ESBL. Report susceptibilities for ESBL producing isolates as recommended in Table 11.4.a.

It is not uncommon for organisms of the EEC group (section 5.5.7) to express an ESBL and a chromosomal inducible cephalosporinase.  Expression of the ESBL can be detected in the usual way (Plate 12.12.B and Plate 12.12.C).

5.5.3. Klebsiella oxytoca and K1 β-lactamase

K. oxytoca produces a chromosomal β‑lactamase known as K1 β-lactamase. Like ESBLs, K1 is inhibited by clavulanic acid but to a lesser degree. Typically, K1 is produced at low (basal) level conferring resistance to ampicillin only with no detectable synergy between clavulanic acid and cephalosporin discs (Plate 12.11.A). In addition to the K1 enzyme, K. oxytoca may also acquire a plasmid mediated ESBL (Plate 12.11.B).

Hyper production of the K1 β-lactamase

Some strains of K. oxytoca hyper-produce the K1 enzyme and have a reduced zone of inhibition of < 6 mm around an amoxycillin-clavulanic acid 60 μg disc. A mild synergy may be observed near a disc containing clavulanic acid (amoxycillin-clavulanic acid or ticarcillin-clavulanic acid) when positioned adjacent to a cefotaxime or ceftriaxone disc (Plate 12.11.C). Greater hyper-production of the K1 enzyme will swamp the weak inhibitory effect of clavulanic acid and the synergy will no longer be visible (Plate 12.11.D).

K1 hyper-producers show a small zone of inhibition around cephalexin 100 μg and cefotaxime 5 μg (Plate 12.11.D). They also have a zone < 6mm in annular radius around ticarcillin-clavulanic acid 85 μg and piperacillin-tazobactam 55 μg discs.

Susceptibilities are reported according to the standard interpretation.

5.5.4. Yersinia enterocolitica

Yersinia enterocolitica susceptibility testing is performed at 30°C on Sensitest Agar using all the criteria applied to members of the Enterobacteriaceae. Although the presence of an inducible cephalosporinase enzyme B (Ambler class C, Bush group 1 β-lactamase) was described in this species, it was shown that this β-lactamase is not highly inducible in the predominant virulent biotype 4, serotype O:38. By contrast, biotype 2 or 3, serotype O:5,27, the second most commonly found bio‑serotype in Australia, produce a highly inducible cephalosporinase enzyme B. Induction of β-lactamase may be demonstrated by the flattening of the zone of inhibition around a cefotaxime 5 μg disc adjacent to an imipenem 10 μg disc. It is advisable to report biotype 2 or 3, serotype O: 5, 27 resistant to penicillins, penicillin/inhibitor combinations and all cephalosporins except cefpirome and cefepime that can be tested.

5.5.5. Inducible cephalosporinases

Further studies on the ESCHAPPM Group.

ESCHAPPM (or ESCAPPM) is a mnemonic for a group of Enterobacteriaceae (Enterobacter cloacae, Enterobacter aerogenes, Serratia marcescens, Citrobacter freundii, Hafnia alvei, Aeromonas hydrophila, Aeromonas caviae, Aeromonas veronii, Providencia stuartii, Providencia rettgeri and Morganella morganii) that are known to produce chromosomal inducible cephalosporinases, inhibited by aztreonam but not by clavulanic acid (AmpC/Bush group 1). Producers of these types of cephalosporinase can be detected in the CDS test by an adjacent disc test (Plate 12.12.A). In the CDS laboratory, we have been looking more closely at members of this group and we have found that as far as AmpC production is concerned ESCHAPPM is not a homogeneous group but consists of 4 distinct subgroups.

The first subgroup comprises E. cloacae complex9, E. aerogenes and C. freundii complex10 and we propose that this subgroup be called the EEC subgroup.  This subgroup gives rise, at a high frequency (10-5 to 10-6), to derepressed mutants that are AmpC enzyme hyper-producers.  This may not always be obvious on disc testing, so that where this enzyme is detected, the strain is reported as resistant to all cephalosporins except the 4th generation irrespective of the zone size. We recommend that for this subgroup, only the results with 4th generation cephalosporins (cefepime and cefpirome) and carbapenems be reported. We consider it unsafe to attempt to interpret the results of testing with other β-lactams.

The second subgroup contains only one species, S. marcescens, which does not give rise to derepressed mutants when exposed to ceftazidime, piperacillin-tazobactam and aztreonam but does so when exposed to other β-lactams including cefotaxime, ticarcillin-clavulanic acid, cefpodoxime, cefotetan, cefoxitin, cefuroxime or cephalexin. With this species, it is considered safe only to report the results of testing of aztreonam, piperacillin-tazobactam and ceftazidime in addition to the 4th generation cephalosporins and carbapenems.

The third subgroup consists of all members of Aeromonas sp. with the exception of A. sobria and A.veronii both of which do not produce the cephalosporinase A1. When exposed to cefotaxime, ticarcillin-clavulanic acid, piperacillin-tazobactam, cefpodoxime, cefotetan, cefoxitin, cefuroxime or cephalexin this subgroup yields AmpC hyper-producing mutants at an extremely variable rate.  Within each species, the rate varies from 10-5 to 10‑8.  On the other hand, we have been unsuccessful in our attempts to obtain any hyper-producing mutants from the same strains by exposure to ceftazidime or aztreonam. Furthermore, when the hyper-producing mutants that are selected by any of the eight β-lactams listed above are tested against ceftazidime or aztreonam, all are susceptible to these two antibiotics. It appears that the substrate specificity of AmpC of Aeromonas sp. is different to that of the AmpC of the EEC subgroup.  Based on these observations, we recommend that it is only safe to report ceftazidime and aztreonam susceptibility as well that of the 4th generation cephalosporins and carbapenems, the latter only in the case of A. caviae (see section 5.2).

The fourth subgroup consists of H. alvei, P. stuartii, P. rettgeri and M. morganii. This group possess an inducible β-lactamase (AmpC) with a high affinity for cephalosporins but it is produced at low levels. Wild strains are resistant only to ampicillin, amoxycillin-clavulanic acid and cephalexin (a characteristic pattern of this subgroup).  Hyper-producers of AmpC are selected only at a low mutation rate of 10-8 when exposed to the β-lactams to which they are susceptible. Members of this subgroup are tested and interpreted by the CDS in the usual way. Where induction is seen, the zone from the edge remote to the flattening is measured and the standard interpretation is applied.

A number of clinical reports stated that infections with susceptible members of this group did not fail therapy with extended spectrum cephalosporins11,12,13. However, the possibility that mutation to hyper-production of AmpC cannot be excluded. Clinical reports should include the comment: although there is little clinical evidence that H. alvei, P. stuartii, P. rettgeri and M. morganii readily fail therapy with extended-spectrum cephalosporins if in vitro susceptibility is proven, resistance can emerge and subsequent isolates should be monitored.

The CDS recommendations on testing and reporting are summarised in Table 11.4.a. (A guide to the reporting of β-lactam antibiotics).

P. vulgaris and P. penneri

P. vulgaris and P. penneri possess a chromosomal inducible cephalosporinase of Ambler class A (Bush group 2e), inhibited by clavulanic acid but not by aztreonam. In the absence of some other resistant mechanism, these organisms are susceptible to amoxycillin-clavulanic acid and derepressed mutants remain susceptible to ceftazidime, cefoxitin and cefotetan. Detection is possible using an adjacent disc testing (flattened zone of CTX 5 µg disc placed near an IPM 10 µg disc) and the susceptibility to AMC 60 µg disc (Plate 12.13.A and Plate 12.13.B).

Citrobacter amalonaticus

Citrobacter koseri and Citrobacter amalonaticus are two species within the genus Citrobacter that have similar biochemical patterns and cannot be differentiated by usual laboratory techniques such as API or Vitek. However, C. amalonaticus produces a chromosomally mediated inducible cephalosporinase of Ambler class A that in many respects resembles that of Proteus vulgaris and Proteus penneri (Plate 12.13.A) while C. koseri lacks this enzyme14. On the CDS plate, a typical pattern of C. amalonaticus is the flattening of the zone of CTX 5 adjacent to the IPM 10 µg disc, a zone > 6 mm around cefepime 10 µg and a borderline AMC 60 µg zone.

Inducible plasmid mediated AmpC

Rare strains of Enterobacteriaceae other than those in the EEC group may acquire a plasmid mediated inducible cephalosporinase equivalent to that possessed by the EEC group15. Such strains show an inhibitory zone < 6 mm around a CTX 5 disc with resistant colonies in the zone. The susceptibilities are reported as for the EEC group.

5.5.6. Metallo‑β‑lactamases (MBLs)

These Ambler class B (Bush group 3) plasmid mediated β‑lactamases hydrolyse penicillins, cephalosporins and carbapenems but not aztreonam. They require zinc ions for enzymatic catalysis and so are inhibited by EDTA (through chelation of Zn++). They are not inhibited by clavulanic acid (Plate 12.9.A).

Screening for MBLs

The MBLs of the Enterobacteriaceae hydrolyse carbapenems less efficiently than they hydrolyse other β-lactams, consequently isolates that express an MBL may still appear susceptible to both imipenem and meropenem. However, their more efficient hydrolysis of other β-lactams and, their non-inhibition by clavulanic acid can be used to screen for their presence (Plate 12.15.A).

Resistance observed with a cefepime 10 μg disc and the absence of a synergistic zone of inhibition between this disc and an adjacent amoxycillin-clavulanic acid 60 μg disc (containing clavulanic acid) is suggestive of the presence of an MBL. Note: Cefepime is not hydrolysed by AmpC β-lactamases.

Confirmation of an MBL

The expression of an MBL can be confirmed by demonstrating the loss of enzymatic activity following chelation of zinc ions. This disc test is performed by positioning a disc loaded with 415 µg of EDTA with its edge 10 mm from the edge of an ertapenem 10 μg, meropenem 5 µg or imipenem 10 μg disc. A zone of growth inhibition between the two discs indicates the presence of an MBL (Plate 12.14.B Pseudomonas and Plate 12.15.B Enterobacteriaceae). Alternatively, confirmation can be achieved by parallel testing of a carbapenem disc and an EDTA supplemented carbapenem disc and the zones of inhibition compared.

Co-expression of MBL and ESBL

It is not uncommon for Enterobacteriaceae isolates to express both an ESBL and an MBL. When using EDTA discs to perform MBL confirmatory testing it is recommended to include an aztreonam 30 µg disc (ATM 30) placed in the centre of the plate.  Aztreonam is not affected by MBL and the isolate will be susceptible unless it also expresses and an ESBL. The co-presence of an MBL and an ESBL can be clearly demonstrated in members of the Enterobacteriaceae by the resistance to ATM 30 and the typical “key hole” observed between ATM 30 and an adjacent AMC 60 disc (plate 12.15.B).

With Pseudomonas aeruginosa, some isolates may show a non-specific synergy between EDTA and a beta-lactam disc including ATM 30 that does not indicate the presence of an MBL.

Note: EDTA discs used in phenotypic detection of MBL are prepared by lining up 6 mm paper discs in an empty Petri dish. Drop 25 µL EDTA 0.05M (Ethylenediaminetetraacetic acid, Sigma, 431788) onto each disc. Let dry for 24 – 48 hour (until dry) with the lid on in 35oC incubator. Transfer discs to a jar and store in a draw.

5.5.7. Klebsiella pneumoniae carbapenemases (KPCs)

Although KPC producing K. pneumoniae have been reported in Europe and USA, the first strain isolated in Australia was reported in September 2010. KPCs are essentially “super” ESBLs of Ambler class A (Bush group 2) plasmid mediated β‑lactamases that hydrolyses all β-lactam antibiotics including the carbapenems. Although inhibited by clavulanic acid and tazobactam, the enzyme is highly potent and affects all β-lactams including the β-lactam/β-lactamase inhibitor combinations including ticarcillin-clavulanic acid, amoxycillin-clavulanic acid and piperacillin-tazobactam (see plates 12.16.A & B).

Due to the low potency discs and the relatively high inoculum used in the CDS test, KPC producers are readily recognised as resistant to all β-lactam antibiotics tested including the carbapenems. The confirmation can be performed phenotypically or by PCR testing.

5.5.8. Salmonella species with decreased susceptibility to ciprofloxacin in systemic infection

Treatment failure has been reported with invasive Salmonella infections where the MIC of ciprofloxacin was ≥ 0.125 mg/L16. On this basis it was recommended that Salmonella species isolated from blood culture should be tested against both nalidixic acid and ciprofloxacin. A single point mutation in the quinolone resistance‑determining region (QRDR) of the topoisomerase gene gyrA in Salmonella species confers resistance to nalidixic acid with an associated decrease in the susceptibility to ciprofloxacin17,18.  Although similar mutations have been described in E. coli and in other members of the Enterobacteriaceae, it is not known if the increase in MIC up to 0.5 mg/L that may result from the mutation has an adverse effect on the clinical response to ciprofloxacin in Gram negative septicaemia. Until more clinical evidence is available we recommend that CDS Users only test Salmonella species causing systemic disease against nalidixic acid as well as ciprofloxacin. The susceptibility to ciprofloxacin is reported as follows:

  • The annular radius of the zone of inhibition is ≥ 6 mm around nalidixic acid 30 μg and ≥ 6 mm around ciprofloxacin 2.5 μg
  1. The MIC of ciprofloxacin is < 0.125 mg/L.
  2. Report as susceptible to ciprofloxacin.
  • The annular radius of the zone of inhibition is < 6 mm (usually 0 mm) around nalidixic acid 30 μg and ≥ 6 mm around ciprofloxacin 2.5 μg
  1. There is a decreased susceptibility to ciprofloxacin with an MIC of ≥ 0.125 mg/L.
  2. Report the susceptibility as follows: “There is decreased susceptibility to ciprofloxacin with the MIC between 0.125 mg/L and 1 mg/L. Treatment failure with ciprofloxacin has been reported with these strains”.
  • The annular radius of the zone of inhibition is < 6 mm (usually 0 mm) around nalidixic acid 30 μg and < 6 mm around ciprofloxacin 2.5 μg

The MIC of ciprofloxacin is > 1 mg/L. Report as resistant to ciprofloxacin

5.5.9. Azithromycin 15 µg disc for the testing of Salmonella enterica subsp. enterica serovar Typhi (Salmonella Typhi) and other Salmonella species isolated from blood culture.

Salmonella Typhi and other Salmonella species isolated from blood culture have been calibrated in the CDS using an azithromycin 15 µg disc. The Australian Antibiotic Guidelines recommend azithromycin as the treatment of choice for Salmonella Typhi.  Although it is surprisingly high we have tentatively accepted a susceptible breakpoint of 16 mg/L as this is based on clinical outcome and in vitro testing reported in the literature19,20. The cut off of the annular radius for susceptible strains is 4 mm. The acceptable range obtained with Escherichia coli ATCC 25922 is 4.7 – 7.2 mm.

5.5.10. Salmonella and gentamicin.

Salmonella are facultative intracellular pathogens. Gentamicin has been shown to be ineffective in killing intracellular Salmonella21.  Do not report gentamicin for Salmonella.

5.5.11. Fosfomycin 200 µg disc for the testing of Enterobacteriaceae from uncomplicated UTI.

This antibiotic has been calibrated for the CDS for use in uncomplicated urinary tract infections using a fosfomycin/trometamol 200µg disc (Oxoid, FOT 200, CT0758B). It is emphasised that this antibiotic has not been calibrated for systemic use and it is best regarded as a urinary antiseptic. Enterobacteriaceae are tested on Sensitest agar. When there is a double zone of confluent growth, the measurement of the annular radius is performed on the inner zone.

 

MIC of susceptible strains                              ≤ 32 mg/L

Annular radius of susceptible strains              ≥ 6 mm

 

Note: With this antibiotic in vitro mutation to resistance is high with the majority of strains tested and this is demonstrated by the presence of resistant colonies within the inhibitory zones on disc testing. However, it is claimed that the reason for the clinical efficacy is that a fosfomycin urine level of > 128 mg/L is maintained for over 24h after a single 3g oral dose22. Acinetobacter species are considered inherently resistant to fosfomycin.

5.6. Haemophilus influenzae and Haemophilus species

Haemophilus influenzae

Susceptibility testing of H. influenzae is performed on Haemophilus Test Medium (section 2.2.1) and incubated as described in section 2.2.4. Some rare strains of H. influenzae grow poorly or not at all on Haemophilus Test Medium.  Susceptibility testing of these strains can be performed on chocolate Columbia Blood Agar (section 2.2.1) and incubated as described in 2.2.4.

It has been described with H. influenzae or H. parainfluenzae that increases in MIC to ampicillin and other β-lactam antibiotics is most likely due to the production of a β-lactamase (TEM type or ROB-1), altered PBPs or in some rare cases a combination of both.  These isolates are referred to as β-lactamase positive amoxicillin-clavulanate resistant (BLPACR) strains. Both TEM type and ROB-1 β-lactamases are class A serine β-lactamases which confer resistance to ampicillin and are inhibited by clavulanic acid.  Their presence can be confirmed using Nitrocefin hydrolysis.

Haemophili with decreased susceptibility to cefotaxime

Isolates of H. influenzae or H. parainfluenzae may have altered PBPs with a lowered affinity for β-lactam antibiotics resulting in resistance to ampicillin and decreased susceptibility to other β-lactam antibiotics especially cephalosporins.  These isolates are referred to as β-lactamase negative ampicillin-resistant (BLNAR) strains23. This decreased susceptibility can be confirmed by susceptibility testing with a cefotaxime or a ceftriaxone 5 μg disc in parallel with the cefotaxime or ceftriaxone 0.5 μg disc. If the inhibitory zone is < 6 mm with the 0.5 μg disc and ≥ 6 mm with the 5 μg disc, report the susceptibility as follows: “There is decreased susceptibility to cefotaxime (or ceftriaxone) with the MIC between 0.5 mg/L and 2.0 mg/L”.

Other Haemophili

Other Haemophilus species, and some H. parainfluenzae isolated from blood culture may not grow on Haemophilus Test Medium.  Susceptibility testing of these organisms is performed on chocolate Columbia Blood Agar (section 2.2.1) and incubated as described in section 2.2.4.

H. aphrophilus and H. paraphrophilus are now included in Aggregatibacter sp. Testing and reporting of these organisms is described in Section 8.

5.7. Helicobacter pylori

A limited range of commonly used antibiotics have been calibrated for susceptibility testing against Helicobacter pylori (Table 11.1.b). The inoculum is prepared in Brain Heart Infusion broth as described in section 2.2.2, subsection ‘Helicobacter pylori’. Susceptibility testing is performed on chocolate Columbia Blood Agar (section 2.2.1) and incubated at 35‑37°C under microaerophilic conditions for 72 hours.

Test no more than 3 antibiotic discs per plate. Discs should be distributed evenly around the plate and no more than 1cm from the edge of the plate. Susceptible strains of H. pylori have large zones of inhibition and these may overlap if discs are positioned any closer than instructed above. Susceptibilities are reported as per the standard interpretation (6 mm).

H. pylori is a difficult organism to work with and dies readily. For this reason, it is not suitable for use as a reference strain for quality assurance testing.

  • Metronidazole 5 μg is tested against Bacteroides fragilis ATCC 25285 on blood Sensitest Agar in an anaerobic atmosphere at 35-37°C for 24 hours (Table 3.b.)
  • Amoxycillin 2 μg, ciprofloxacin 2.5 μg, erythromycin 5 μg, rifampicin 5 μg and tetracycline 10 μg are tested against aureus ATCC 9144 on Sensitest Agar in air at 35°C for 24 hours (Table 11.3.a).

5.8. Pasteurella species

The CDS method has now been extended to include other Pasteurella species (P. gallinarum, P. pneumotropica and Mannheimia haemolytica (previously Pasteurella haemolytica) in addition to P. multocida. Some strains require CO2 to grow and it is, therefore, now recommended that susceptibility testing of Pasteurella species be performed on blood Sensitest Agar (Section 2.2.1) at 35‑37°C, in an atmosphere of 5% CO2. Ampicillin, benzylpenicillin, erythromycin, ciprofloxacin, moxifloxacin, marbofloxacin and tetracycline have been calibrated for Pasteurella species (Table 11.1.b).

5.8.1. Ampicillin and benzylpenicillin susceptibility

Pasteurella multocida

P. multocida should be tested against ampicillin 5 μg. Do not use benzylpenicillin 0.5 u. An inhibitory zone with an annular radius of ≥ 6 mm around the ampicillin 5 μg disc indicates susceptibility to benzylpenicillin.

Pasteurella species (other than P. multocida)

Two antibiotic discs, benzylpenicillin 0.5 u and ampicillin 5 μg are used for testing and reporting the susceptibility to benzylpenicillin, ampicillin, amoxycillin and ceftiofur. Categories of susceptibility are defined as follows:

  • Susceptible to benzylpenicillin, ampicillin, amoxycillin and ceftiofur.

The annular radius of the inhibitory zone around benzylpenicillin 0.5 u is ≥ 4 mm.

  • Reduced susceptibility to benzylpenicillin, ampicillin, amoxycillin and ceftiofur.

The annular radius of the inhibitory zone around benzylpenicillin 0.5 u is < 4 mm but ≥ 6 mm around ampicillin 5 μg.

Report as: “There is decreased susceptibility to benzylpenicillin, ampicillin, amoxycillin and ceftiofur with the MIC between 0.25 and 2.0 mg/L.”

  • Resistant to benzylpenicillin, ampicillin, amoxycillin and ceftiofur.

The annular radius of the inhibitory zone around ampicillin 5 μg is < 6 mm.

5.9. Pseudomonas aeruginosa

P. aeruginosa possesses an inducible chromosomal AmpC (Bush group 1) β‑lactamase. However, the rate of mutation to resistance is low (approximately 10-9), unlike members of the Enterobacteriaceae. In vivo, P. aeruginosa is unlikely to give rise to hyperproducing mutants except in sequestered sites (cystic fibrosis, osteomyelitis24).

P. aeruginosa may also acquire an ESBL. Double disc testing with a ticarcillin-clavulanic acid disc (contains clavulanic acid) adjacent to a ceftazidime disc, facilitates detection of these ESBLs (Plate 12.14.A).

An MBL, (carbapenemase, Ambler class B or Bush group 3) has been found in some strains of P. aeruginosa. Pigmented MBL producing P. aeruginosa isolates are highly resistant to all β-lactams with no zone of inhibition except aztreonam.  See section 5.5.8. for additional information on the nature and detection of these enzymes (Plate 12.14.B) and Power Point ASM 2011. When performing the detection of MBL, be aware that with some P. aeruginosa, EDTA may show non-specific synergy with all antibiotics discs including aztreonam and non β-lactam antibiotic discs. Therefore, it is necessary to always include an ATM 30 disc in MBL confirmatory test.

GES β-lactamases: Multidrug resistant P. aeruginosa has been associated with GES-2. Although rare, GES enzymes have been identified worldwide. The GES enzyme (Guiana extended spectrum) is an Ambler class A extended spectrum β-lactamase, first described in 200021. They are plasmid encoded and have a broad hydrolysis spectrum including penicillin, extended spectrum cephalosporins and may extend to cephamycins as well as imipenem. Isolates displaying carbapenem resistance and that have a negative disc approximation test with EDTA (see 5.5.8.) may possess a GES enzyme.

5.9.1. Fosfomycin 200 µg disc for the testing of Pseudomonas species from uncomplicated UTI.

This antibiotic has been calibrated for the CDS for use in uncomplicated urinary tract infections using a fosfomycin/trometamol 200 µg disc (Oxoid, FOT 200, CT0758B). It is emphasised that this antibiotic has not been calibrated for systemic use and it is best regarded as a urinary antiseptic. Pseudomonas sp. are tested on Sensitest agar.

MIC of susceptible strains                              ≤ 32 mg/L

Annular radius of susceptible strains              ≥ 6 mm

Note: With this antibiotic in vitro mutation to resistance is high with the majority of strains tested and this is demonstrated by the presence of resistant colonies within the inhibitory zones on disc testing. However, it is claimed that the reason for the clinical efficacy is that a fosfomycin urine level of > 128 mg/L is maintained for over 24h after a single 3g oral dose. When there is a double zone of confluent growth, the measurement of the annular radius is performed on the inner zone.

5.10. Stenotrophomonas maltophilia

Most wild strains of S. maltophilia are usually susceptible to sulphonamide and resistant to trimethoprim, but show a marked synergy between the two antibiotics. A pear shape or comet tail zone of inhibition between trimethoprim-sulfamethoxazole (sulphonamide component) and trimethoprim is typical and indicates synergy between the two antibiotics (Plate 12.17.A). However, rare strains of S. maltophilia may be resistant to sulphonamide as well as trimethoprim and therefore appear resistant to trimethoprim-sulfamethoxazole (SXT 25) (Plate 12.17.B).

NOTE: S. maltophilia usually have a hazy or light growth visible within the SXT 25 inhibitory zone. This does not imply resistance to trimethoprim-sulfamethoxazole or sulphamethoxazole. Repeat the test using a 1/10 dilution of the CDS inoculum. The inhibitory zone will then be more distinctive and easier to read.

No zone of inhibition around an imipenem (or meropenem) disc and the marked synergy between trimethoprim and trimethoprim-sulfamethoxazole suggests that the isolate is likely to be S. maltophilia (Plate 12.17.A).

Although some isolates of S. maltophilia appear to be susceptible to aminoglycosides at 35°C, the MICs of aminoglycosides recorded at 30°C with these strains were 32 or 64 fold higher than those recorded at 35°C. Therefore, in the CDS test S. maltophilia is considered to be resistant to all aminoglycosides

S. maltophilia possesses two chromosomal β-lactamases – L1, a penicillinase/carbapenemase inhibited by EDTA and L2, a cephalosporinase inhibited by clavulanic acid. There is a high rate of mutation (10-4 to 10-6) leading to resistance to β-lactams, aminoglycosides and quinolones and should be considered resistant to all antibiotics intended for use as monotherapy.

The drug of choice for the treatment of infections caused by S. maltophilia is trimethoprim-sulfamethoxazole.

Although we recommend that S. maltophilia be reported resistant to all β-lactam antibiotics (Table 11.4), this can lead to a therapeutic dilemma when the isolate is resistant to sulphonamides (Plate 12.17.B) or the patient is allergic to sulphonamides. In these difficult, uncommon situations, test aztreonam, ceftazidime, ticarcillin-clavulanic acid, piperacillin-tazobactam, ciprofloxacin and moxifloxacin using the criteria set out for Pseudomonas species (susceptibility equals an annular radius of ≥ 6 mm). A warning such as “A combination of antibiotics is necessary for successful therapy” should then be issued with the susceptibility report.

 

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