11/13/22

Bacilos de Gram negativo

não fermentadores, aeróbios
Oportunistas

In 2019, six pathogens were each responsible for more than 250 000 deaths
associated with AMR (figure): E coli, Staphylococcus aureus, K pneumoniae, S
pneumoniae, Acinetobacter baumannii, and Pseudomonas aeruginosa, by order of
number of deaths

Resistencia a antibióticos

The Lancet 2022 399629-655DOI: (10.1016/S0140-6736(21)02724-0)

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Bad Bugs, No Drugs: No ESKAPE!

Enterococcus faecium
Staphylococcus aureus
Klebsiella pneumoniae
Acinetobacter baumannii
Pseudomonas aeruginosa
Enterobacter/ E. coli

Bacilos de Gram negativo não fermentadores, aeróbios

oportunistas
Degradam vários carbohidratos

– Pseudomonas
• > 190 espécies. P. aeruginosa; P. fluorescens; P. putida: P. Oxidase +
stutzeri; P. alcaligenes
– Burkholderia
• > 30 espécies: B. cepacia complex; B. pseudomallei; B. mallei

– Stenotrophomonas maltophilia

– Acinetobacter Não Flagelada Oxidase -

• > 43 espécies. A. baumannii; A. lwoffii; A. hemolyticus; A. pitti,
A. nosocomialis

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Habitat/Contaminação
Dispersas no ambiente (solo e água)
– Pseudomonas
• P. aeruginosa- sol. Desinfectantes, gotas oculares, fluidos diálise e
equipamentos; banhos em hidroterapia, sol. de lentes de contacto;
raramente parte da flora humana normal

– Burkholderia
• B. cepacia complex; -coloniza doentes c/ fibrose cística
• B. pseudomallei- - Regiões tropicais e subtropicais
• Inalação ou por contacto da pele lesionada com solo ou água
– Stenotrophomonas maltophilia
• Coloniza frequentemente secreções de doentes (secreções
respiratórias; urina, exsudados de feridas, Soluções desinfectantes;
Equipamento respiração; jarras de flores;

– Acinetobacter
• Colonização de doentes em ambiente hospitalar frequente. Commonly
colonizes skin, oropharynx secretions, respiratory secretions, and urine

Frequente em dispositicvos médicos e superfícies em ambiente

Manifestações clínicas
• Pseudomonas
P. aeruginosa- Adquiridas em ambiente hospitalar: Inf. Trato
resp. inferior; ITU; Inf. de feridas; Bacterémia. Inf. Respiratórias em
fibrose cística (maioria das crianças c/ 10 anos são colonizadas; a
resposta inclui inflamação, com exacerbação de problemas
respiratórios
Adquiridas na comunidade em não imunocomprometidos: Otite
externa; Foliculite; Inf. Oculares; Endocardites; Inf. Respiratórias em
fibrose cística; UTI em doentes c/ cateter.

• Burkholderia
• B. cepacia complex; -Inf. respiratórias em doentes com
fibrose cística; ITU (especialmente em cateterizados),
septicémia

• B. pseudomallei- Melioidose - Inf. Assintomática; inf.

Crónica (semelhante a inf. por M. tuberculosis) ou sepsis
fulminante
• Factores de risco: diabetes, insuf. Renal ou talassemia

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Stenotrophomonas maltophilia
•Bacteremia, meningite; Conjuntivites; Endocardites; Pneumonia;
Infecções da pele; Inf. Trato urinário (especialmente em cateterizados);
Importante patogénico nosocomial

Acinetobacter
• Pneumonia; Inf. da pele e tecidos moles; ITU (especialmente em
cateterizados); Meningite secundária; bacterémia.

• Mostly concern critically ill patients in intensive care units

(ICUs): increased lenght of hospital stay; antibiotics;
mechanical ventilation
Factores de risco:
• Hospitalização (hospitalização prolongada; ventilação
mecânica)
• Comprometimento das defesas do organismo;
• Tratamento prolongado com antibióticos de largo espectro;

P. aeruginosa

• Crescimento a 42oC
• Utilização de citrato
• Produção de pigmentos, ex:
– Piocianina- : azul
– Pioverdina: amarelo
– Piorubina vermelho-acastanhado
• Cheiro a maçãs
• Produção de energia por via oxidativa; pode utilizer
nitratos como aceitadores de electrões
• Utilizam vários compostos orgânicos c/o fonte de
carbono e azoto

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Critical traits contributing to the pathogenicity

of P. aeruginosa

• Versatilidade metabólica
• Produção de vários fatores de virulência
• Formação de biofilmes
• Resistência a antimicrobianos

Gram - virulence features presented in previous class also important

P. aeruginosa
Virulência
• Cápsula
• Endotoxina
• Toxinas e enzimas
• Exotoxina (Exotoxina A, S e T)
• Exotoxina A (ETA) – mais importante factor de virulência
• Bloqueia a síntese proteica ao bloquer o elongamento da cadeia peptídica nas células eucariotas (semelhança
com a toxina diftérica)
• Parece contribuir para a dematonecrose nas feridas de queimados, lesões da cornea em inf. Oculares e lesões
tecidulares nas inf. Pulmonares crónicas.
• Actividade imunossupressiva
• Piocianina (oxide e reduz difrentes moléculas-destroi microrganismos competidores e células de humanos);
pioverdina- sideróforo
• Elastase
• Ramnolipido
• Fosfolipase C
• Protease alcalina
• Flagelos
• Formação de biofilme
• Resistência a antibióticos

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Acute and chronic infections

P. aeruginosa

Alginate, also referred to as

mucoid exopolysaccharide (MEP),
is the most studied
Exopolyssacharide and the major
component of mucoid P.
aeruginosa biofilms.

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Ramnolipido

• Glicolipido produzido por P. aeruginosa

• Surfactante bacteriano
• Vantagens da sua produção
– Actividade antimicrobiana e antifúngica- anti-Serratia marcescens, Klebsiella
pneumoniae, Staphylococcus aureus and Bacillus subtilis
– Virulência- associado com a deterioração dos doentes com pneumonia
associada ao ventilador e infeções da cornea.
• Integram-se na membrana epitelial celular

Aquisição de virulência e resistência a antimicrobianos

Botelho J., Grosso F., Quinteira S., Mabrouk A.,
Peixe L.
J Antimicrob Chemother 2017;
pJB37 megaplasmid (446 kb) and comp doi:10.1093/jac/dkx143
arison with similar plasmid backbones

Tn6356, a novel blaVIM-2-harbouring transposon

ter (tellurite resistance) operon

Biogenesis and mechanical

function of type IV pili (T4P),
pil (pili) operon twitching motility
and biofilm formation
che (chemotaxis) operon

required for flagella mediated

chemotaxis

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Exame cultural de P. aeruginosa

Crescem em diferentes meios culturais

Pesquisa de P. aeruginosa- Cetrimida Agar

• Cetrimida- é um dos compostos de amónio quaternário
presentes (Brometo de Alquiltrimetilamónio; HTAB; CTAB;
CTABr)

• Bactericida em Gram+ e alguns Gram-

• Cloreto de Mg e Sulfato K favorecem a produção dos

pigmentos pioverdina e piocianina

Águas de piscinas
Análise cosméticos, desinfectantes

Outras espécies com Importância

Clínica
• Pseudomonas fluorescens

• P. putida

• P. stutzeri

• P. alcaligenes
• ………

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Intrinsic resistance in non-fermentative gram-negative bacteria

Generally intrinsically resistant to benzylpenicillin, first- and second-generation
cephalosporins, glycopeptides, lipoglycopeptides, fusidic acid, macrolides, lincosamides,
streptogramins, rifampicin and oxazolidinones

X X X X X X X X X X
No inhibition of class B carbapenemases Ceftolozane+ tazobactam
Ceftazidime + avibactam
1 Acinetobacter baum annii m ay appear to be susceptible to am picillin-sulbactam due to activity of sulbactam with this species.
2 Acinetobacter is intrinsically resistant to tetracycline and doxycycline but not to m inocycline and tigecycline. Aztreonam/avibactam
3 Burkholderia cepacia com plex includes different species. Som e strains m ay appear susceptible to som e beta-lactam s in vitro but they are clinically resistant.
4 Burkholderia cepacia and Stenotrophomonas maltophilia are intrinsically resistant to all am inoglycosides. Intrinsic resistance is attributed to poor perm eability and putative efflux. In
addition, m ost Stenotrophomonas maltophilia produce the AAC(6’)Iz enzym e.
5 Pseudomonas aeruginosa is intrinsically resistant to kanam ycin and neom ycin due to low level APH(3’)-IIb activity.
6 Stenotrophomonas maltophilia is typically susceptible to trim ethoprim -sulfam ethoxazole, but resistant to trim ethoprim alone.
7 Stenotrophomonas maltophilia is intrinsically resistant to tetracycline but not to doxycycline, m inocycline and tigecycline.

Mechanisms of intrinsic antibiotic

resistance in P. aeruginosa
Main mechanisms leading to intrinsic
resistance to antibiotics:
• outer-membrane permeability
• efflux systems
• production of antibiotic-inactivating
enzymes ex. blaAmpC; blaoxa

Main mechanisms leading to acquired

resistance to antibiotics:
• outer-membrane permeability • Quinolones and β-lactams penetrate cell
membranes through porin channels.
• efflux systems (e.g. overexpression) • Aminoglycosides and polymyxins promote
• Acquisition of antibiotic-inactivating their own uptake by interacting with LPS
enzymes ex. blaVIM; on the outer membrane.

MexABOprM efflux pump

(overexpression leads to meropenem resistance)

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P. aeruginosa – ‘High Risk Clones’

Biofilm
Production
ST111 – VIM-2
ST235 – VIM-2,
GES-6
ST175 – VIM-2 Pigment
Reduced
production Chronic Infection Motility
ST244 – VIM-2 decrease

Mutation frequency
increase

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VIM-2 (SPA, GER)

VIM-20 (SPA)
IMP-1 (JAP) VIM-1 (CRO, GRE,
VIM-2 IMP-22 (SPA) GERM, PHI)
(SPA) VIM-2 (EUR, ASIA)
VIM-4 (EUR, IRA)
IMP-6 (KOR, CHI,
JAP)
IMP-7 (SIN, JAP)
VIM-2 (IC, JAP, NDM-1 (ITA)
CHI) KPC-2 (COL)
IMP-1 (JAP) GES-5 (RUS, SPA)
IMP-6 (CHI) GES-6 (POR)
IMP-10 (JAP)

VIM-1 (SPA,
GRE)
VIM-2 (EUR,
CAN, VEN,
IND)
VIM-4 (HUN,
GRE)
KPC-2 (COL)
IMP-7 (CZR)
IMP-13 (FRA)
IMP-18 (CAN)

Antibioterapia
Stenotrophomonas maltophilia
Naturalmente resistente a vários antibióticos, incluindo carbapenemos
(produtor de carbapenemase).

Naturalmente sensível a:
l Trimetoprim-sulfametoxazole
l Cloranfenicol; Tetraciclina
l Ceftazidima

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Intrinsic resistance in non-fermentative gram-negative bacteria

Generally intrinsically resistant to benzylpenicillin, first- and second-generation
cephalosporins, glycopeptides, lipoglycopeptides, fusidic acid, macrolides, lincosamides,
streptogramins, rifampicin and oxazolidinones

X X X X
X X

1 Acinetobacter baum annii m ay appear to be susceptible to am picillin-sulbactam due to activity of sulbactam with this species.
2 Acinetobacter is intrinsically resistant to tetracycline and doxycycline but not to m inocycline and tigecycline.
3 Burkholderia cepacia com plex includes different species. Som e strains m ay appear susceptible to som e beta-lactam s in vitro but they are clinically resistant.
4 Burkholderia cepacia and Stenotrophomonas maltophilia are intrinsically resistant to all am inoglycosides. Intrinsic resistance is attributed to poor perm eability and putative efflux. In
addition, m ost Stenotrophomonas maltophilia produce the AAC(6’)Iz enzym e.
5 Pseudomonas aeruginosa is intrinsically resistant to kanam ycin and neom ycin due to low level APH(3’)-IIb activity.
6 Stenotrophomonas maltophilia is typically susceptible to trim ethoprim -sulfam ethoxazole, but resistant to trim ethoprim alone.
7 Stenotrophomonas maltophilia is intrinsically resistant to tetracycline but not to doxycycline, m inocycline and tigecycline.

Antibioterapia
Acinetobacter baumannii
• Naturalmente suscetível a vários antibióticos
• Frequentemente, opções terapêuticas limitadas-aumento das
Resistências
Carbapenemos
Sulbactam
Polimixinas (Colistina)
Aminoglicosídeos (Amicacina)
Glicilciclinas (Tigeciclina)
Rifampicina
Tetraciclinas (Minociclina)

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Intrinsic resistance in non-fermentative gram-negative bacteria

Generally intrinsically resistant to benzylpenicillin, first- and second-generation
cephalosporins, glycopeptides, lipoglycopeptides, fusidic acid, macrolides, lincosamides,
streptogramins, rifampicin and oxazolidinones

X X X X

X X X X
X X

1 Acinetobacter baum annii m ay appear to be susceptible to am picillin-sulbactam due to activity of sulbactam with this species.
2 Acinetobacter is intrinsically resistant to tetracycline and doxycycline but not to m inocycline and tigecycline.
3 Burkholderia cepacia com plex includes different species. Som e strains m ay appear susceptible to som e beta-lactam s in vitro but they are clinically resistant.
4 Burkholderia cepacia and Stenotrophomonas maltophilia are intrinsically resistant to all am inoglycosides. Intrinsic resistance is attributed to poor perm eability and putative efflux. In
addition, m ost Stenotrophomonas maltophilia produce the AAC(6’)Iz enzym e.
5 Pseudomonas aeruginosa is intrinsically resistant to kanam ycin and neom ycin due to low level APH(3’)-IIb activity.
6 Stenotrophomonas maltophilia is typically susceptible to trim ethoprim -sulfam ethoxazole, but resistant to trim ethoprim alone.
7 Stenotrophomonas maltophilia is intrinsically resistant to tetracycline but not to doxycycline, m inocycline and tigecycline.

Multi-drug Resistant A. baumannii

¡ Carbapenems Increasingly compromised

1. Porin loss or modification (CarO)

2. Modification of penicillin-binding proteins (PBPs)
3. Acquired β-lactamases

Metallo-β-lactamases Carbapenem hydrolysing oxacillinases (CHDLs)

• IMP
• OXA-23-like
• VIM
• OXA-40-like
• SIM
• OXA-58-like
• NDM

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Intrinsic resistance in non-fermentative gram-negative bacteria

Generally intrinsically resistant to benzylpenicillin, first- and second-generation
cephalosporins, glycopeptides, lipoglycopeptides, fusidic acid, macrolides, lincosamides,
streptogramins, rifampicin and oxazolidinones

X X X X X X X X X

X X X X
X X

1 Acinetobacter baum annii m ay appear to be susceptible to am picillin-sulbactam due to activity of sulbactam with this species.
2 Acinetobacter is intrinsically resistant to tetracycline and doxycycline but not to m inocycline and tigecycline.
3 Burkholderia cepacia com plex includes different species. Som e strains m ay appear susceptible to som e beta-lactam s in vitro but they are clinically resistant.
4 Burkholderia cepacia and Stenotrophomonas maltophilia are intrinsically resistant to all am inoglycosides. Intrinsic resistance is attributed to poor perm eability and putative efflux. In
addition, m ost Stenotrophomonas maltophilia produce the AAC(6’)Iz enzym e.
5 Pseudomonas aeruginosa is intrinsically resistant to kanam ycin and neom ycin due to low level APH(3’)-IIb activity.
6 Stenotrophomonas maltophilia is typically susceptible to trim ethoprim -sulfam ethoxazole, but resistant to trim ethoprim alone.
7 Stenotrophomonas maltophilia is intrinsically resistant to tetracycline but not to doxycycline, m inocycline and tigecycline.

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Four A. baumannii isolates were recovered from a

37-year-old woman with a history of alcohol
abuse, admitted, in 2006, in the emergency ward
of the Hospital S. Teoto´nio, Viseu, Portugal, with
acute necrohaemorrhagic pancreatitis. Therapy
was initiated with meropenem and fluconazole on
the day of admission. Nine days after meropenem
administration, Enterococcus faecium,
Enterococcus casseliflavus and A. baumannii
(HST1) isolates were recovered from
intraperitoneal fluid. Another A. baumannii isolate
(HST1a) was obtained from blood samples,
revealing an identical susceptibility profile to that
of HST1, including resistance to all available
antibiotics, except amikacin. Therapy was
replaced by a combination of ampicillin and
amikacin, for 19 days, maintaining prophylaxis
with fluconazole. Nine days after changing
therapy a pancreatic abscess was drained.
Cultures of the intraperitoneal fluid revealed A.
baumannii colonies (HST2 and HST3) presenting
Journal of Hospital Infection 75 (2010) 73–84 not only an MDR pattern similar to that of HST1,
but also resistance to amikacin (Table I).

Acinetobacter baumannii dynamics in our hospitals

<2001 2003 2005 2007 2009 2011 2013 2015

ST98
ST208
ST103 ST218

Sporadic STs
Virulence factors Antibiotic Resistance
Clones CHDL Others
CPS hem O ompA, bap, csuA/ABCDE, bfmRS, pgaABCD, plcC/plcD, CARBA-R AM G-R Other-R
lpsB/lpxC, basABCDEFGHIJ/barAB/bauABCDEF, entA

ST98 OXA-40 - ✚✚✚ ✚ ✚

✚✚
Fucosamine strA, strB tet(B), sul1

ST103 OXA-58 ✚✚ ✚✚✚ ✚

✚ ✚
Fucosamine sul2

ST208 OXA-23 Pseudaminic ✚✚ ✚✚✚ ✚✚✚ ✚✚ ✚✚

Tn2006 acid
✚✚✚
ST218 OXA-23 Legionaminic ✚✚✚ ✚✚✚ ✚✚✚
✚✚✚ msr(E), mph(E), sul1,
Tn2006 acid armA, AMEs aac(6´)Ib-cr, catB8
✚✚✚
ST218 OXA-23 Legionaminic
✚✚✚ ✚✚✚ ✚ ✚
✚✚✚
(H482) Tn2006 acid (Extra strA, strB tet(B)
lysine-Wzc)
-
Sporadic OXA-40 - ✚✚ ✚ - - -
STs (n=1)

Silva L. et al, 2021; Grosso F. et al, 2010, 2011

Legionamic and pseudodaminic acid- derivatives of sialic acid- molecular mimicry of host glycans

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