Major groups of β-lactamases in Gram-negative bacteria 
Klebsiella pneumoniae is a nosocomial pathogen commonly implicated in hospital outbreaks with a propensity for antimicrobial resistance towards mainstay β-lactam antibiotics and multiple other antibiotic classes. The successful proliferation, transmission and infection of the Gram-negative bacterium can be attributed to a myriad of factors including host factors, environmental factors, virulence factors and a large repertoire of antibiotic resistance mechanisms. The poor treatment outcomes and limited treatment options are consequences of the successful pathogenesis and spread of antibiotic resistance in the increasingly common β-lactamase producing K. pneumoniae bacterium. The review briefly explores the biology, successful pathogenesis and antibiotic resistance of K. pneumoniae as well as the detection and characterisation techniques of important strains.
- Klebsiella pneumoniae
- Antibiotic resistance
The evolution of the Gram-negative bacillus in an era of antibiotic use has resulted in a changed epidemiology, wherein
Several mechanisms contribute towards antimicrobial resistance and virulence in Gram-negative bacteria and may even work in concert to achieve multidrug resistance profiles [7–8]. Resistance determinants usually mediate resistance by inactivating the antimicrobial agent, modifying the antibiotic or its target and decreasing antimicrobial drug concentrations within the cell [9–11]. A common form of enzymatic inactivation of antibiotics is the acquisition and expression of β-lactamase genes within bacterial species, such as
Characterisation of clinically relevant
Once established in the hospital setting, the proliferation and spread of MDR strains can occur within and between hospitals . The molecular characterisation of β-lactamases and the molecular typing of
2. Epidemiology of multidrug-resistant
Broad-spectrum β-lactamases initially emerged in
Hospital outbreaks of ESBL-producing bacteria, particularly
Carbapenem resistance in
3. Classification of
K. pneumoniae isolates
A study conducted by Drancourt and collegues (2001) aimed at re-establishing and confirming the taxonomy of the genus
4. General characteristics of
K. pneumoniae bacteria
4.1. Culture and metabolic characteristics
Useful tests in determining enterobacterial taxonomy include carbon source utilisation tests, glucose oxidation test in the presence or absence of pyrroloquinoline quinone, gluconate- and 2-ketogluconate dehydrogenase tests and tetathionate reductase and β-xylosidase tests . All
4.2. Genomic characteristics
Microbial pathogens are capable of modifying inherent virulence or patterns of spread through evolutionary processes, which can often be mediated by HGT . The acquisition of pathogenicity islands and virulence plasmids are mechanisms by which
5. Virulence factors and the role in pathogenesis of
The significant impact of
The prerequisite to an infection is often the mucosal pathogen’s ability to adhere [7,73].
Surface saccharides that have been associated with
Finally, the growth of
Virulence genes typically researched include
6. Clinical manifestations of
K. pneumoniae infections
Unlike their Gram-positive counterparts, invasive infections and metastatic spread are rare for extra-intestinal Gram-negative pathogens, such as
The virulence factors expressed could contribute to the range of clinical manifestations of infections, but the geographical restriction of certain manifestations could alternately be dependent on host factors typical to that region [26,27,89]. Host factors could include the frequency of diabetes mellitus, genetic predilections, underlying prevalent diseases, alcoholism, socioeconomic determinants and the availability of quality healthcare [26,27,89,90].
7. Treatment of
K. pneumoniae infections
Appropriate therapeutic options are often determined based on the antibacterial spectrum, convenience of use and tolerability of antimicrobials, such as third- and fourth-generation cephalosporins. The factors influencing appropriate antimicrobial treatment are also dependent on local bacterial susceptibility patterns and patient risk profiles, which may ultimately determine the risk of infection with opportunistic and potentially antibiotic-resistant pathogens . Multidrug-resistant bacterial strains, such as
7.1. Treatment of multidrug-resistant
K. pneumoniae infections
The global emergence of multidrug-resistant Gram-negative bacilli is an unprecedented problem, which is exacerbated by the focus on improving existing classes of drugs instead of developing new classes of drugs with alternate targets over the past 50 years [4,5]. The rise in the rate of multidrug-resistant bacteria and the increasingly limited treatment options is exemplified by ever-prevalent ESBL-producing
Typical characteristics of ESBL-producing members of the
In a retrospective study conducted by Micek and colleagues (2010), a better outcome was believed to be associated with correct initial combination antimicrobial therapy when empirically treating Gram-negative bacteria-mediated sepsis as compared to monotherapy . In the aforementioned study, a combination of a antipseudomonal fluoroquinolone, such as ciprofloxacin, or an aminoglycoside with a carbapenem (imipenem and meropenem), piperacillin-tazobactam or cefepime as initial treatment for severe Gram-negative bacterial infections offered a broader spectrum of activity . Additional retrospective studies further favour combination therapy in CRE infections for which treatment options have been reduced mainly to colistin, tigecycline, some aminoglycosides and fosfomycin [4,18]. Although fosfomycin appears active
8. Antibiotic-resistance mechanisms in
K. pneumoniae isolates
Innate antimicrobial susceptibility could be impacted by adaptive responses, resulting in alterations to gene expression and cell physiology, which are induced in response to the pathogen’s natural environmental stresses or within a host [10,103–105] Three modes of antibiotic resistance existing in bacteria, such as
Changes in membrane permeability and drug flux can be influenced by variable expression and regulation of the efflux pumps . Within the
Bacterial cells can exist as single cells, the planktonic form, or within communities drawn together by a self-produced biopolymer matrix and attached to a surface [46,105,115,116]. The latter is referred to as a biofilm and confers survival advantages in the form of improved resistance to host immune defences, resistance to biocides, increased resistance to antimicrobial compounds and higher plasmid transfer rates within that environment, which could include antibiotic resistance genes [10,46,75,115,116]. Genetic elements conferring potential resistance genes are easily transferred horizontally both intra- and interspecies due to the close genetic resemblance between bacteria of the
Finally, resistance towards β-lactam antibiotics are mainly mediated by β-lactamase enzyme production, which is capable of hydrolysing third-generation cephalosporins and monobactams [48,58,107,121,122]. Other factors at play besides ESBL production include cases of ESBL hyperproduction due to promoter upregulation after direct mutation, inserted transposable elements in close proximity to the promoter and the capacity of a strain to coproduce more than one ESBL .
9. Classification of β-lactamases
Enzyme-mediated resistance to β-lactam antibiotics was initially discovered in
|1||C||Cephalosporinase||Penicillins, cephalosporins, carbapenems*, monobactams*|
|2b||A||Penicillinase||Penicillins, early cephalosporins, β-lactamase inhibitor combinations*|
|2be||A||Extended-spectrum β-lactamase||Penicillins, cephalosporins, monobactams, β-lactamase inhibitor combinations|
|2d||D||Cloxacillinase||Penicillins (including oxacillin and cloxacillin)|
|2df||D||Carbapenemase||Carbapenems and other β-lactams|
|2f||A||Carbapenemase||All current β-lactams|
|3||B||Metallo-β-lactamase||All β-lactams, except monobactams|
Ambler molecular classes A, C and D enzymes typically possess serine within the active site, while class B enzymes contain zinc [6,37,38,51]. Nine structural/evolutionary families have been described during the classification of ESBL variants . The variants include Belgium extended-spectrum β-lactamase (BEL), Brazilian extended-spectrum β-lactamase (BES), CTX-M, Guyana extended-spectrum β-lactamase (GES), oxacillinase (OXA),
Three definitions of ESBLs have been proposed, which include a classical definition, a broadened definition and an all-inclusive definition . The classical definition originally defined an ESBL as derivatives of broad-spectrum TEM and SHV enzymes and later more functionally defined as β-lactamases of the Ambler class A or functional group 2be capable of hydrolysing extended-spectrum cephalosporins and monobactams, while still being inhibited by β-lactamase inhibitors and poorly hydrolysing cephamycins and carbapenems . The classical definition did not, on the other hand, account for the β-lactamases with similar hydrolysis profiles and dissimilar evolutionary backgrounds, such as CTX-M, GES and VEB enzymes . A broader definition by Livermore (2008), included TEM and SHV variants with weaker ESBL activity, the enzymes with similar hydrolysis but dissimilar sources, as well as β-lactamases possessing wider resistance to the parent types that do not fall within the 2be functional group (e.g. OXA variants and AmpC type mutants). The wider resistance observed is to oxyimino-cephalosporins . Lee and colleagues (2012) have independently extended the broadened definition of ESBLs to include AmpC ESBLs from the Ambler class C; thus designating ESBLs as: aESBLs, cESBLs and dESBLs . The broadened definition is limited in that ESBLs with concurrent carbapenem and oxyimino-cephalosporin resistance are excluded .
Finally, the all-inclusive definition classifies ESBLs into three classes: ESBLA (class A ESBLs), ESBLSM (miscellaneous ESBLs including as AmpC and OXA-type ESBLs) and ESBLCARBA (β-lactamases encompassing ESBLs with carbapenem hydrolysing activity) [94,128]. The GES-1 β-lactamase, for example, has hydrolysis profiles resembling that of other ESBLs, but six GES β-lactamases have illustrated carbapenemase activity, being GES-2, -4, -5, -6, -11 and-14 . Bush and colleagues (2009), on the other hand, felt the term ESBLCARBA as clinically confusing as ESBLs should be treatable with carbapenems and should thus remain more accurately classified as carbapenemases . Bush and colleagues (2009) further disputes the definitions set by Giske (2009) by stating that AmpC-producers although treatable with carbapenems may develop resistance easily and should thus not be classified together with ESBLs [128,130]. The all-inclusive definition thus further excludes the clinical criteria in which ESBLs should have sensitivity to available β-lactamase inhibitors and current definitions of ESBLs, AmpC β-lactamases and carbapenemases should be kept independent . The most common ESBL-encoding genes detected include SHV-, TEM- and CTX-M-type enzymes .
10. Risk factors for ESBL-producing
K. pneumoniae infections
The clinical outcomes of inadequate empirical treatment with broad-spectrum antibiotics with no activity against the isolated causative bacterium (
Generalised factors in at-risk patients commonly include severe illness, underlying medical conditions, recent surgery, haemodialysis, multiple or excessive antibiotic use, the use of medical devices, such as lines and tubes, prolonged hospitalisation, ICU admittance, admittance at long-term health facilities or nursing homes and international travel to endemic areas . An important risk factor in modern society is the risk of acquiring ESBL-producing
The clinical manifestation of disease can be attributed to numerous host-dependent factors, which may range geographically but it is also influenced by socioeconomic determinants and the quality of healthcare at hand [26,27,89,90]. Underlying complications or illness that may result in an increased risk of
11. Spread, prevention and control
The rise in antimicrobial-resistance among bacteria, such as those described as ‘ESKAPE’ pathogens (
Factors impacting the spread and control of MDR bacteria include spread of plasmids and are impacted by the food chain or international travel . During the travels, acquisition can occur in the absence of healthcare contact or along with leisure and medical tourism [45,137]. In healthcare settings, overcrowding is a key factor in exacerbating the faecal–oral route of transmission by either direct or indirect contact by healthcare workers . The contact that staff have with patients during unassuming social interactions, such as taking a patient’s blood pressure and the touching of inanimate objects in the patient’s environment, could contribute to horizontal spread of pathogens, especially when elective hand hygiene practices are neglected [4,138]. The implementation of alcohol-based hand rubs and regular educational programmes are thus important steps in control measures undertaken . The role of post-acute care facilities in dissemination of MDR bacteria is also stressed by Perez and colleagues (2010) .
Infection control measures undertaken can include: (i) increased barrier precautions, (ii) isolation of infected patients, (iii) appropriate antibiotic treatment duration and (iv) epidemiological standards for the handling of equipment as well as patient wounds [4,14,59]. A method investigated for its potential to reduce cross-contamination and infection rates in clinical settings, such as the ICU, is the effect of selective digestive tract decontamination (SDD) for the elimination of cephalosporin-resistant
Several key shortcomings have, however, been identified by the World Health Organization (WHO) in the combat against antimicrobial resistance . The issues are discussed under four topics which include: (i) lack of commitment and data, (ii) unconfirmed drug quality and irrational use, (iii) poor prevention and control of infections and (iv) languishing research into new antimicrobial agents and tools, including diagnostic tests and antimicrobials . The resulting policy package recommended by the WHO thus firstly suggests that governments adopt and finance comprehensive national plans with accountability and engaging civil society by creating public awareness . The second recommendation is based on improving surveillance and laboratory capacities, whilst the third advises local governments to guarantee an uninterrupted supply of essential, quality-assured medication . The regulation and promotion of the correct use of former-mentioned medication is also emphasised along with good patient care . Finally, the last two recommendations involve improvement of infection prevention and control while encouraging research and development of new tools, including diagnostic tests and antimicrobials .
12. Laboratory diagnosis of β-lactamase producing
K. pneumoniae isolates
In light of increasing antibiotic resistance among bacteria, surveillance of drug-resistance patterns within clinical settings and clinically relevant pathogens is significant particularly when deciding on appropriate treatment for complicated infections . The detection of ESBL-producing bacteria requires tests that can accurately discern between ESBL producers and bacteria possessing alternative resistance mechanisms, such as inhibitor-resistant-β-lactamases, cephalosporinase overproduction and SHV-1 hyperproduction .
12.1. Biochemical and phenotypic detection techniques
Characteristics associated with ESBL-producing
Initially, the DDST following methodology specified by the Clinical and Laboratory Standards Institute (CLSI) guidelines was intended for the differentiation between ESBL-producing
Cloxacillin has been added to agar media for the inactivation of cephalosporinases, an AmpC β-lactamase, whereas both clavulanate and EDTA have been added when MBLs are produced concurrently with ESBLs for the latter’s identification and confirmation . The detection of extended spectrum Ambler class D OXAs is, on the other hand, complicated due to weak inhibition and no inhibition observed towards clavulanate and EDTA, respectively [47,149]. A unique characteristic attributed to most class D β-lactamases, including OXA-48-type enzymes, is the inhibition of activity by sodium chloride (NaCl)
Carbapenemases can, on the other hand, also be screened for in at-risk patients using selective media, such as CHROMagar KPC medium (CHROMagar Ltd, France), BrillianceTM CRE medium (Thermo scientific, UK) and SUPERCARBA medium . Typically, methods of detecting carbapenemases make use of inhibition tests utilising boronic acid, clavulanic acid, EDTA and tazobactam [112,127]. Carbapenemase resistance in
12.2. Automated detection of ESBLs
Automated systems used for the detection of ESBLs are the VITEK®2 ESBL test (bioMérieux, France) and the Phoenix ESBL test (Becton Dickinson, USA), both of which monitor the bacterial growth response to expanded-spectrum cephalosporins [14,47]. The VITEK®2 ESBL test (bioMérieux, France) consists of cards with wells, whereas the automated Phoenix ESBL test (Becton Dickinson Biosciences, USA) consists of five wells containing a cephalosporin with or without clavulanic acid . Another method that could be used for the detection of β-lactamase and carbapenemase activity is the matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometry (MS), which analyse carbapenem molecule hydrolysis, although its efficiency in detecting OXA-48 producers remains uncertain [127,152–154].
12.3. Newer detection methods
Molecular investigations of outbreaks can be complicated when spurred by the spread of highly mobile plasmids . Antimicrobial resistance genes are often carried on varied plasmids, which have been implicated in MDR Gram-negative bacteria outbreaks, as illustrated in a study by Tofteland and colleagues (2013), wherein the
13. Typing of
K. pneumoniae isolates
Genetic typing of
The PFGE molecular method is highly discriminatory and is the gold standard typing method in the characterisation of
14. Commonly characterised
K. pneumoniae strains
Sequence typing has allowed for the characterisation of
Antibiotic resistance is often discussed in terms of selection and subsequent proliferation of MDR strains or the horizontal transfer of genetic elements encoding resistance, such as plasmids . A combination of proteomics and molecular techniques could thus be used for the characterisation of plasmids within outbreak
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