Gram negative bacilli can be resistant to various beta lactams, ranging from penicillins to carbapenems. Such resistance may be constitutive or acquired. Constitutive or in-built resistance is typically due to restricted entry of antibiotics into cells, by "gating" of entry points called porins, which span the outer hydrophobic cell wall in gram negative bacilli. The more hostile an environment the bacterium lives in, the more impermeable will be the porins. Thus, porins in Acintobacter, which lives in liquid media such as irrigation solutions in hospitals, is 100 times less permeable than E.coli, which lives in the relatively friendly and protected environment of the gut with a host of other Enterobacteriaceae. Here, it is worth mentioning that Acintobacter and Pseudomonas, albeit being gram negative bacilli, are not from the same family as Enterobacteria. That is to say, unlike E.coli, Klebsiealla, Citrobacter, Providentia, Serratia, Enterobacter and Proteus species, Pseudomonas and Acintobacter do not live in the gut.
Although not important for beta-lactams, which act in the peri-plasmic space between the inner and outer membranes, antibiotic resistance in gram negative bacteria is also served by an efflux pump, which simply pumps out intracellular antibiotics to the exterior. This mechanism is obviously more important for antibiotics which act inside the cell, such as aminoglycosides, quinolones, tetracyclines and macrolides.
Resistance can also be acquired ecologically, from other successful bacteria, for example in the gut. The most important example of this is the so called "accessory genome" epitomised by plasmids.
Some plasmids are so successful that they can self transmit during bacterial reproduction. They are called constitutive plasmids. Plasmids have their own genetic code called replicons. Plasmids from the same group have similar replicons, and are incompatible with each other. When two similar plasmids are in the same cell, they will only multiply once the cell has divided and the competing plasmids have passed into separate daughter cells.
Constitutive plasmids cause the bacterium in which they reside to become "addicted" to them. Such "plasmid addiction" often involves a toxin-antitoxin system. The toxin tends to be stable while the anti-toxin is unstable and subserved by the plasmid. When the plasmid is lost, so is the anti-toxin, resulting in destruction of the bacterium by the intracellular toxin. In this way, the plasmid ensures that only those bacteria that carry it, survive.
The Ambler Classification for gram-negative resistance to beta-lactams is widely used and comprises four classes of beta-lactam resistance.
Class A- Comprises both broad spectrum beta-lactamases encoded by genes such as TEM and SHV and ESBLs, such as CTX-M-15 (Cefotaxime hydrolysing, first described in Munich). These group of bacteria are sensitive under lab and clinical conditions to inhibition by beta-lactamase inhibitors such as clavulanate, sulbactam, tazobactam and avibactam.
Class A also includes carbapenamases (CPEs) such as KPC (Klebsiella pneumoniae carbapenamase).
Class B- Comprises MBLs or bacteria producing metallo-beta-lactamases, most of which contain zinc as the "metal". Examples include imipenamase (IMP), Verona-integron encoded metallo beta lactamase (VIM), and the New Delhi Metallo-beta-lactamase (NDM). These group of bacteria will hydrolyse almost all beta lactams except aztreonam (a monobactam). They are relatively insensitive to beta lactamase inhibitors.
Class C- These group of bacteria are called Amp-C (or ampicillin hydrolysing). Amp-C bacteria produce cephalosporinases that hydrolyse 1st, 2nd and 3rd generation cephalosporins. Their ability to hydrolyse 2nd gen cephalosporins, called cefamycins, comprising cefoxitin and cefotetam, sets them apart from ESBLs, which cannot hydrolyse cefamycins. This latter property is used in the lab to differentiate Amp-C from ESBL. Amp-C are also insensitive to clavulanate.
Class D- called OXA or oxacillinases. These bacteria are not inhibited by clavulanate either. They hydrolyse 3rd gen cephalosporins but with a differential between cefotaxime and ceftazidime.
There are various ways of screening for beta-lactamases. Paricularly important is to detect ESBL or carbapenamase producing strains, as their spread will lead to widespread bacterial resistance. Isolation methods are often used for such patients.
The two most widely used are the Combined Disc test and the modified Hodge test.
The combined disc test is a variation of the Double Disc Synergy Test (DDST).
Here, two separate 30 ug discs containing the bacterial inoculum are placed 30 mm from each other (centre to centre) in agar. One disc contains cefotaxime, while the other contains amoxycillin-clavulanate. For ESBL producing bacteria (typically strains of E.coli, K.pneumoniae, K.oxytoca or Proteus species), there will be synergy between cefotaxime and clavulanate and the area of bacterial lysis in the cefotaxime disc will be greater where it apposes the clavulanate disc. (Remember, ESBLs as well as carbapenamases belong to Ambler Class A, which is the only class of beta-lactamases significantly inhibited by clavulanate). The appearance of the cefotaxime disc therefore takes on the shape of a keyhole when ESBL or carbapenamase is present.
The problem arises when there are other classes of beta lactamases that hydrolyse 3rd gen cephalosporins, thus masking the presence of ESBL. It's still important to pick up ESBL due to epidemiologic and clinical reasons. When ESBL is being co-produced with significant amounts of Amp-C (Ambler Class C), the classical synergy with clavulanate may not be seen, as Amp-C is not inhibited by clavulanate. This limitation can be overcome by bringing the discs closer than 30 mm, or by adding cloxacillin to the agar, as this inhibits Amp-C.
When ESBL is being co-produced with Ambler Class B, the metallo-beta-lactamases, it is possible to neutralise the latter with EDTA (chelates Zinc), thus bringing out the synergy with the clavulanate disc.
Unfortunately, no tests exist, except PCR, to tell apart the co-existence of ESBL and Ambler Class D- OXA.
Although not important for beta-lactams, which act in the peri-plasmic space between the inner and outer membranes, antibiotic resistance in gram negative bacteria is also served by an efflux pump, which simply pumps out intracellular antibiotics to the exterior. This mechanism is obviously more important for antibiotics which act inside the cell, such as aminoglycosides, quinolones, tetracyclines and macrolides.
Resistance can also be acquired ecologically, from other successful bacteria, for example in the gut. The most important example of this is the so called "accessory genome" epitomised by plasmids.
Some plasmids are so successful that they can self transmit during bacterial reproduction. They are called constitutive plasmids. Plasmids have their own genetic code called replicons. Plasmids from the same group have similar replicons, and are incompatible with each other. When two similar plasmids are in the same cell, they will only multiply once the cell has divided and the competing plasmids have passed into separate daughter cells.
Constitutive plasmids cause the bacterium in which they reside to become "addicted" to them. Such "plasmid addiction" often involves a toxin-antitoxin system. The toxin tends to be stable while the anti-toxin is unstable and subserved by the plasmid. When the plasmid is lost, so is the anti-toxin, resulting in destruction of the bacterium by the intracellular toxin. In this way, the plasmid ensures that only those bacteria that carry it, survive.
The Ambler Classification for gram-negative resistance to beta-lactams is widely used and comprises four classes of beta-lactam resistance.
Class A- Comprises both broad spectrum beta-lactamases encoded by genes such as TEM and SHV and ESBLs, such as CTX-M-15 (Cefotaxime hydrolysing, first described in Munich). These group of bacteria are sensitive under lab and clinical conditions to inhibition by beta-lactamase inhibitors such as clavulanate, sulbactam, tazobactam and avibactam.
Class A also includes carbapenamases (CPEs) such as KPC (Klebsiella pneumoniae carbapenamase).
Class B- Comprises MBLs or bacteria producing metallo-beta-lactamases, most of which contain zinc as the "metal". Examples include imipenamase (IMP), Verona-integron encoded metallo beta lactamase (VIM), and the New Delhi Metallo-beta-lactamase (NDM). These group of bacteria will hydrolyse almost all beta lactams except aztreonam (a monobactam). They are relatively insensitive to beta lactamase inhibitors.
Class C- These group of bacteria are called Amp-C (or ampicillin hydrolysing). Amp-C bacteria produce cephalosporinases that hydrolyse 1st, 2nd and 3rd generation cephalosporins. Their ability to hydrolyse 2nd gen cephalosporins, called cefamycins, comprising cefoxitin and cefotetam, sets them apart from ESBLs, which cannot hydrolyse cefamycins. This latter property is used in the lab to differentiate Amp-C from ESBL. Amp-C are also insensitive to clavulanate.
Class D- called OXA or oxacillinases. These bacteria are not inhibited by clavulanate either. They hydrolyse 3rd gen cephalosporins but with a differential between cefotaxime and ceftazidime.
There are various ways of screening for beta-lactamases. Paricularly important is to detect ESBL or carbapenamase producing strains, as their spread will lead to widespread bacterial resistance. Isolation methods are often used for such patients.
The two most widely used are the Combined Disc test and the modified Hodge test.
The combined disc test is a variation of the Double Disc Synergy Test (DDST).
Here, two separate 30 ug discs containing the bacterial inoculum are placed 30 mm from each other (centre to centre) in agar. One disc contains cefotaxime, while the other contains amoxycillin-clavulanate. For ESBL producing bacteria (typically strains of E.coli, K.pneumoniae, K.oxytoca or Proteus species), there will be synergy between cefotaxime and clavulanate and the area of bacterial lysis in the cefotaxime disc will be greater where it apposes the clavulanate disc. (Remember, ESBLs as well as carbapenamases belong to Ambler Class A, which is the only class of beta-lactamases significantly inhibited by clavulanate). The appearance of the cefotaxime disc therefore takes on the shape of a keyhole when ESBL or carbapenamase is present.
The problem arises when there are other classes of beta lactamases that hydrolyse 3rd gen cephalosporins, thus masking the presence of ESBL. It's still important to pick up ESBL due to epidemiologic and clinical reasons. When ESBL is being co-produced with significant amounts of Amp-C (Ambler Class C), the classical synergy with clavulanate may not be seen, as Amp-C is not inhibited by clavulanate. This limitation can be overcome by bringing the discs closer than 30 mm, or by adding cloxacillin to the agar, as this inhibits Amp-C.
When ESBL is being co-produced with Ambler Class B, the metallo-beta-lactamases, it is possible to neutralise the latter with EDTA (chelates Zinc), thus bringing out the synergy with the clavulanate disc.
Unfortunately, no tests exist, except PCR, to tell apart the co-existence of ESBL and Ambler Class D- OXA.
