Though the topic of resistant Gram-negative organisms has come up frequently in the past (eg. all the repetitive ESCAPPM questions), the college have never really asked about the extreme end of the drug resistance spectrum until Question 20 from the first paper of 2018, where the resistance profile of a Klebsiella species comes up as "R" for everything. All the listed drugs are ineffective, according to the antimicrobial susceptibility profile. What enzymes could be responsible for this, the college asked, and, more importantly,  "how would you manage this clinical scenario?"

In general the topic appears to be valuable because these organisms are appearing more and more frequently in the Australian ICU environment and because the CDC/ECDC keep using terms like "crisis," "catastrophic consequences" and "nightmare scenario" to describe the problem of emergent drug resistance (Zilahi et al, 2016). It is clearly useful to have a toolkit of standard strategies for this situation.

The time-poor exam candidate will likely want to limit their reading to this excellent review by Pop-Vicas and Opal (2013), which has a good discussion fo resistance mechanisms and enzymes. For management recommendations, one of the better contemporary articles is the Joint Working Party statement by Hawkey et al (2018)

What do you mean, "multi-resistant"?

The term itself is poorly defined. How many drugs do you have to be resistant to before you are multi-resistant? Apparently the European CDC defines it as "resistant to three or more antibiotic classes", whereas if the bug is susceptible to only one or two classes, it gets called "extremely" or "extensively" drug resistant (XDR). Organisms which are not susceptible to any of the known agents were defined as "pandrug-resistant" (PDR). Magiorakos et al (2011) describe the ECDC's attempt to create these definitions. Obvious there is a lot of disagreement among experts, including on serious matters (such as what one might consider as a "class" of antibiotics, and how to group them). As a hilarious side note, it is important to point out that the ECDC is the European Centre for Disease Prevention and Control, whereas the American counterpart is the Center for Disease Control and Prevention. That aside, the definitions seem to be widely accepted. They are based on in vitro susceptibility testing, and the Magiorakos article is remarkable because it has excellent tables with lists of bacteria which have intrinsic resistance to certain antibiotics, i.e. all Citrobacter freundii organisms are born with the innate ability to survive just about any concentration of cefazolin. 

Mechanisms of extended spectrum antibiotic resistance

Question 20 asked specifically which enzymes are responsible for the drug resistance profile, strongly implying that the college only wanted to hear the keywords "β-lactamase" or "carbapenemase", even though the Klebsiella strain they gave the candidates was also resistant to aminoglycosides, fluoroquinolones, tetracyclines and cotrimoxazole. As such, the resistance mechanisms to all of the aforementioned drugs will be discussed here, even though the college clearly wasn't interested in that level of depth. The examiners awarded this part of Question 20 with only 10% of the marks, which suggests that it was viewed as something largely irrelevant. As such, the topic will not be subjected to the dissection it deserves (as it is fascinating). The level of depth resembles what is offered in Pop-Vicas and Opal (2013), which is the source of most of the following references.

β-lactam cephalosporin and carbapenem resistance  occurs as the result of β-lactamase enzymes. At least one of the resistance enzyme in Klebsiella from the SAQ is a carbapenemase, potentially either KPC or New Delhi metallo-beta-lactamase (NDM-1). β-lactamase enzymes are classified according to a system called the Bush-Jacoby-Medeiros classification:

  • Class A: resistance to monobactams, third-generation cephalosporins, 
    but these are inhibited by clavulanate and tazobactam, at least in vitro. To this class belongs KPC, the Klebsielle pneumoniae carbapenemase in Question 20 from the first paper of 2018.
  • Class B: resistance to penicillins, cephalosporins and carbapenems. 
    NOT inhibited by clavulanate and tazobactam. These are the ones known as "metallo-beta-lactamases", because their active site contains a divalent metal cation, usually zink. This is the group to which belongs the NDM-1 enzyme.
  • Class C: resistance to cephalosporins; limited inhibition by clavulanate and tazobactam. These are the enzymes induced in ESCAPPM organisms
  • Class D: "Oxacillinase"; resistance to carbapenems is slightly inhibited by clavulanate. These are also known as OXA  β-lactamase enzymes.

Cotrimoxazole resistance develops when the bacteria either change the structure of their dihydrofolate reductase enzyme (DHFR), or produce copious quantities of it, or both. In the latter case, cotrimoxazole becomes totally ineffective. This is not new. Even dated articles (eg. Huovinen, 1995) report isolates of Gram negatives which have extreme cotrimoxazole resistance (MIC in the range of 1g/L) because they synthesise two hundred times the normal amount of a heavily mutated DHFR. 

Fluoroquinolone resistance occurs when the drug targets mutate. These targets are DNA gyrase and DNA topoisomerase IV enzymes, the genes for which have key vulnerabilities in the unimaginatively named "quinolone resistance determining regions" (QRDRs). Additionally, E.coli and P.aeruginosa routinely express drug efflux pumps (Piddock, 1999). Both the target susceptibility to the drug and the intracellular concentration decrease.

Aminoglycoside resistance  occurs either when the bacteria synthesise inactivating enzymes, or when the ribosomal drug target mutates. When the inactivating enzymes are genereated, they digest and destroy the aminoglycoside molecules through phosphorylation adenylylation or acetylation. This is something which will be very drug-specific as all aminoglycoside molecules are structurally dissimilar, with unexpected effects. For instance, a bug may be resistant to hardcore antibiotics like amikacin, but retain sensitivity to "softcore" options like gentamicin because the affinity of the enzyme for gentamicin is lower.

On the other hand, if the 16S subunit of the ribosome changes in some way (usually by methylation of the 16S rRNA drug target by methylaze enzymes), the changed target confers high-grade resistance to all aminoglycosides, even those in the novel drug development pipeline. Doi & Arakawa (2007) note that these methylase enzymes resemble those found in actinomycete fungi such as Streptomyces, organisms which secrete aminoglycosides and which are therefore intrinsically immune to their effects.

Tetracycline resistance develops when bacteria change the binding site on their ribosomes, or express efficient efflux pumps. The efflux pumps might be drug-specific and saturable, but binding site mutations confer high-grade resistance to all tetracyclines, which usually also means tigecycline. Apart from these mechanisms, there are also tetracycline-specific ribosomal protection proteins (which gently encourage the drug to dissociate off the ribosome) and tetracycline-inactivating enzymes, creatively group-named "tetracycline destructases" (Grossman, 2016). In short, multiple teracycline resistance mechanisms may be active simultaneously.

Approaches to multidrug-resistant Gram negative infection

The following generic strategies can be recommended:

Decide: do you even need to treat?  The sputum sample in Question 20 from the first paper of 2018 had a "light growth" of Klebsiella. Is it really the pathogen which has made the patient sick?

This happens all the time. Consider: a patient has been in the ICU for weeks. All kinds of broad-spectrum agents have been thrown at them, and they eventually survived their catastrophic event, now slowly weaning off the ventilator via a tracheostomy. They have a fever one day and the registrars dutifully culture their sputum. Horrendomonas sp. is cultured, and found to be multi-resistant. At this stage, confronted with the question "should we start horrendopenem", the pragmatic intensivist will retort with profanities, pointing out that there is no clinical evidence that the patient is systemically unwell from this organism, and that after weeks of broad-spectrum fusillade one would not expect normal respiratory flora to grow on the plates, as Horrendomonas and fungi is all that remains in this patient's blasted Dresden-like microbiome.

Control the source. This is good advice in any situation, but particularly where there is little expected benefit from antibiotics. Thus, remove the biofilm by chest physiotherapy or by removing the IDC/ central line, drain that abscess, et cetera. Microorganisms may be up to 1000 times more resistant to antibiotics when they form a biofilm, as compared to their "planktonic" form.

Ask for help. The college answer to Question 20 from the first paper of 2018 calls for a "specialist ID opinion", in a rare acknowledgement of the expertise of another specialty. Indeed the panresistant situation they describe probably merits an approach beyond looking up the recommendations in the Sanford guide. What will the infectious diseases specialist do, that the intensivist cannot? Likely their access to extended drug susceptibility data will be better than yours, and their experience with unusual antibiotics and unusual dosing regimens will be greater. 

Options for antimicrobial therapy

The line of depressing susceptibility findings should not discourage the intensivist from pursuing antibiotic therapy. One merely needs to get a little creative.

Dosing to MIC

Just because the lab reports an "R" does not immediately mean that the agent is going to have zero in-vivo activity. For instance, the microbe may be "R" to gentamicin, but gentamicin will be excreted in the urine, where it will reach a concentration hundreds of times in excess of MIC, thereby retaining good activity. This means that in vitro sensitivity profiles may not always be reflected in the clinical response to antibiotics, particularly where the infection is in a particularly susceptible environment. Another example of such an environment is the CSF  (where you might like to give the antibiotics directly into the EVD).

 The other natural implication of this is that increasing the dose of the drug will defeat the resistance. This may well be true. Pea et al (2017) used meropenem to treat carbapenemase-producing ESBLs successfully by simply using much more meropenem. Whereas a 2g tds (6g/day) dose already seems excessive in most normal circumstances, the authors threw caution to the wind and gave their patients humongous doses up to 13.2g/day, as continuous infusions (using meropenem levels as a guide) with a median treatment length of 14 days. In case anybody ever wonders how much meropenem is a safe amount, this study gives a good answer. The authors did not report as to whether the patients were growing meropenem crystals in their urine.

Exotic agents of antimicrobial therapy

The laboratory does not routinely test for all antimicrobial drugs. There are some which the infectious diseases pharmacist keeps at the back of the antimicrobial pantry, for just these sorts of situations. The following list was compiled after trawling through numerous articles, and should not be viewed as a firm recommendation - this is merely the spectrum of infrequently used agents which might still have activity. The best single reference for novel antibiotics (ones which are on their way towards the market) is probably  Zilahi et al (2016).

  • Monobactams
    • Aztreonam
  • β-lactams
    • Temocillin
    • Mecillinam or pivmecillinam (oral)
  • Cephalosporin and extended spectrum β-lactamase inhibitor:
    • Ceftazidime-avibactam
    • Ceftolozane-tazobactam
  • Carbapenems with and without carbapenemase inhibitor:
    • Meropenem-vaborbactam
    • Tebipenem (oral)
    • Faropenem (oral)
  • Tetracyclines
    • Tigecycline
  • Fluorocyclines
    • Eravacycline
  • Polymyxins
    • Colistin (polymyxin E)
    • Polymyxin B, whichis more toxic
  • Aminoglycosides
    • Amikacin
    • Plazomicin
  • Aminocyclitols
    • Streptomycin
    • Spectinomycin
  • Phosphonic acid derivatives
    • Fosfomycin

Antibiotic synergy for extremely resistant Gram-negatives

An excellent article by Kalan & Wright (2011) describes some of the possible synergies between antibiotics. The article was not specifically discussing resistant Gram-negatives, but the salient features applicable to them have been summarised below. The college in their answer mention that "recommendation is combination antimicrobial therapy" but do not specify whose recommendation that is. Presumably they were not referring to Hawkey et al (2018), as that statement came out approximately two weeks before the written paper. The authors admit that "most of the current evidence for the advantage of combination therapy ...derives from observational studies and reports". It is not totally clear that combination therapy is better than monotherapy, and studies tend to come up with wildly different contradictory conclusion, thereby generating some very confused systematic reviews (eg. Paul et al, 2014)

Recommended combinations include the following cocktails:

  • For KPC strains, colistin + meropenem + tigecyline or ceftazidime/avibactam
  • For OXA strains, aztreonam or ceftazidime/avibactam as monotherapy
  • For metallo-β-carbapenemase producers, colistin + fosfomycin, +/- tigecycline 

Add colistin to anything. The polymyxin will interfere with the bacteral membrane, making it more permeable to the second agent. This is partiocularly effective if the main reason for drug failure is the presence of efficient efflux pumps or a decreased affinity of the drug for the mutant target. Colistin, trimethoprim and sulfonamides (Simmons, 1970) seems to be a winning combination. Similarly, colistin, tigecycline and a carbapenem (even if there is carbapenemase) works well, at least on the level of case series.

Add gentamicin to anything - occasionally, the AAC(60)-Ib enzyme mutation which confers resistance to amikacin spares gentamicin (Gonzalez-Padilla et al, 2014

Use two drugs from the same class. Combination of carbapenems (eg. ertapenem and doripenem together) seems to work for highly resistant carbapenemase producers. Cecarelli et al (2013) found that ertapenem is such a high-affinity substrate for carbapenemase that it could act as a decoy, distracting the enzyme and allowing another carbapenem to do its work. 

Exotic approaches

These are well described by Nigam et al (2014), and because the strategies are highly experimental nobody should mention them in an actual exam answer. They are included here mainly for interest and completeness.

Monoclonal antibodies: for example, anti-PcrV: a monoclonal Fab fragment against the type-III secretion system of Pseudomonas,  (Milla et al, 2014). This blocks the secretion of bacterial toxin. 

Pilicides: for example, ZFH04269, which inhibits FimH the type 1 fimbria adhesin, thereby interefering with ability of E.coli to colonise anything (Totiska et al, 2013)

Bacteriophage therapy is coming back in vogue (Matsuzaki et al, 2005); the bheaviour of the virus and its "predator-prey" killing knetics tend to favour the use of this therapy for decontamination of biofilms with a low colonisation count (i.e. you would not rely on it to treat a raging sepsis).

Bacteriocins are peptides secreted by bacteria to inhibit the growth of other species in the normal course of dog-eat-dog competition which characterises the poor, nasty, brutish lifestyle of microorganisms. Cleveland et al (2001) describes their use in food preservation. Their disadvantages include the potential for resistance (after all it's just another antibiotic) and the fact that as peptides, they are rapidly degraded by peptidases.

Killing factors are also secreted by bacteria, but this time to kill neighbouring members of their own species during a time of colony starvation, in effect a form of bacterial cannibalism. So far their use has been largely experimental and theoretical (Liu et al, 2010)

Antibiotic effects of non-antibiotic drugs is something to consider- many substances have antibiotic effects in addition to their other "proper" effects. For example, barbiturates, NSAIDs, PPIs, diuretics and beta-blockers all  have some antimicrobial effects (Cederlund & Mårdh, 1993). Specific mentions could be made; for example amiloride seems to have some good effect on Gram-positive organisms, propanolol kills S.aureus, aspirin kills Klebsiella and phenothiazine antipsychotics seem to possess broad-spectrum activity against both Gram-positie and Gram-negative organisms. At present no published data exists on on the clinical microbicidal effects of tricyclic antidepressants at above-MIC concentrations (apparently, 150mg/L for amitryptiline), as the management of those patients generally tends to focus on rescuing them from near-lethal tricyclic toxicity. 

Prevention of spread

Though the use of gloves and handwashing is not as sexy as resurrecting antibiotic classes from the 1950s, these are important in controlling the spread of multiresistant Gram-negatives, and this is reflected in the college answer to Question 20 from the first paper of 2018, where half of the answer is dedicated to infection control procedures. These are discussed in greater detail in the chapter on Strategies to prevent the transmission of multi-resistant organisms. They are largely generic, i.e. the strategies used to prevent the spread of an MDR Klebsiella are identical to those one might use for MRSA or VRE. Below, the list of suggestions is modified from the discussion section of Question 12 from the first paper of 2016.

Infection control:

Active surveillance cultures

  • Identifies the "reservoir" for spread
  • Allows precautions to be cost-effectively focused on the reservoir.
  • The health care workers themselves may become colonised reservoirs.
  • Performed on all patients, on admission, and then periodically (eg. weekly).
  • In facilities found to have a high prevalence on initial sampling, a facility-wide culture survey is indicated.

Contact precautions

  • Hand hygiene
    • Soap and water for visibly contaminated hands
    • Alcohol-based rub for routine pre-and-post-contact hygiene
    • Monitoring of compliance should be performed
  • Disposable gloves
  • Disposable gowns
  • Patients colonised by MROs may be cohorted together.
  • Contaminated areas should be identified by obvious cautonary signs

Eradication of existing colonies

  • Effective treatment of existing cases
  • Decontamination of colonised health care workers
  • Environmental disinfection
  • Routine disinfection of equipment between patient contacts
  • Wherever possible, individualised equipment for every patient

Organisation-level changes to improve infection control

Organisation

  • Infection control specialty team, composed of ICU specialists, infectious diseases specialists, senior nursing staff, laboratory staff and administration staff.
  • Allocated resources to MRO surveillance, compliance monitoring and education
  • "Champions" - staff allocated to promote the existing policies and monitor adherence

Education

  • "Widespread campaign", as recommended by the SAQ model answer
  • Awareness-rasing posters
  • Lunchtime meetings
  • Promulgated reading material
  • Education bundle as a part of mandatory employee training

Data collection and audit

  • Data collection and MRO colonisation record
    • Records of MRO results allow colonised patients to be identified early
  • Regular review of collected information, resistance and transmission patterns
  • Regular audit of the efficacy of implemented strategies
  • Regular comparison of policies to those of peer hospitals
  • Regular reevaluation and amendment of infection control policies

References

Nordmann, Patrice, Gaelle Cuzon, and Thierry Naas. "The real threat of Klebsiella pneumoniae carbapenemase-producing bacteria." The Lancet infectious diseases 9.4 (2009): 228-236.

Hawkey, Peter M., et al. "Treatment of infections caused by multidrug-resistant Gram-negative bacteria: report of the British Society for Antimicrobial Chemotherapy/healthcare Infection Society/british Infection Association Joint Working Party." Journal of Antimicrobial Chemotherapy 73.suppl_3 (2018): iii2-iii78.

MacVane, Shawn H. "Antimicrobial resistance in the intensive care unit: a focus on gram-negative bacterial infections." Journal of intensive care medicine 32.1 (2017): 25-37.

Schneider, Elena K., et al. "Antibiotic–non-antibiotic combinations for combating extremely drug-resistant Gram-negative ‘superbugs’." Essays in biochemistry 61.1 (2017): 115-125.

Simmons, N. A. "Colistin, sulphamethoxazole, and trimethoprim in synergy against Gram-negative bacteria." Journal of clinical pathology 23.9 (1970): 757-764.

Pea, Federico, et al. "Might real-time pharmacokinetic/pharmacodynamic optimisation of high-dose continuous-infusion meropenem improve clinical cure in infections caused by KPC-producing Klebsiella pneumoniae?." International journal of antimicrobial agents 49.2 (2017): 255-258.

Ceccarelli, Giancarlo, et al. "Successful ertapenem-doripenem combination treatment of bacteremic ventilator-associated pneumonia due to colistin-resistant KPC-producing Klebsiella pneumoniae." Antimicrobial agents and chemotherapy 57.6 (2013): 2900-2901.

Gonzalez-Padilla, Marcelino, et al. "Gentamicin therapy for sepsis due to carbapenem-resistant and colistin-resistant Klebsiella pneumoniae." Journal of Antimicrobial Chemotherapy 70.3 (2014): 905-913.

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