1. Final upgrading: Extreme-drug-resistant (XDR) Acinetobacter baumannii carrying blaOXA-23 in a patient with acute necrohaemorrhagic pancreatitis
Luísa Vieira Peixe
REQUIMTE. Laboratório de Microbiologia, Faculdade de Farmácia, Universidade do Porto, Portugal

2. REQUIMTE- Microbiology Research Group Goals Goals
Epidemiology study of antibiotic resistant bacteria in order to implement containment measures, methodologies for their detection, and design of new compounds
Hospitals
Food and animal production environment
Humans community
Environment
VRE, Enterobacteriaceae- PL/TEM-52; PL/CTX-M-15, Pseudomonas-VIM
Enterococcus gentaR,
Tn1546
PL/CTX-M-14; PL/CTX-M-15 (Enterobacteriaceae)
VRE-CC17
Acinetobacter OXA-23
Salmonella-intI1 OXA-30, sul3 Enterobacteriaceae- PL/TEM-52
Salmonella-intI1MDR, Enterobacteriaceae- PL/TEM-52
Strategies
Characterization of bacteria, genes and mobile genetic elements from different ecological niches
AAC :1001
JAC (In press)
AAC :1545
CMI :1131
JAC :297
CMI :1047 JAC :1139
EID :1985
AAC :836
AAC : AAC :451
JAC :1370
AAC :3613
AEM :3743
AAC :2140
AEM :3364
CMI :755
AAC :3220
Main Achievements
(1) Emergence and International dissemination of MDR strains: VRE, ESBL-producing Enterobacteriaceae, Carbapenemase-producing Pseudomonas and Acinetobacter, Salmonella MDR
(2) New genetic structures carrying antibiotic resistance genes driving co-selection and maintenance of resistant bacteria, e.g., In100
(3) Interchange of genetic elements/bacteria between different ecological niches
Food producing animals and food of animal origin are a reservoir of clinically relevant clones/resistance genes
Worrisome contribution of hospital sewage for aquatic environmental contamination by resistant bacteria
We Evidenced:

3. Acinetobacter baumannii - Emergent Pathogen
Acinetobacter on the news!
Although a plethora
(some would say a surfeit1) of immunological
and molecular techniques is available for the
discrimination of pathogen isolates, many aspects
of pathogen diversity remain poorly described and
incompletely understood. One of the microrganisms that catch our atention is Acinetobacter, mainly resulting from the clinical relevance of this genus in the last decade.

4. Acinetobacter baumannii - Emergent Pathogen
Significant nosocomial pathogen
Especially in immunocompromised patients or with underlying diseases
Mainly Pneumonia (also bacteremia, meningitis, skin and wound infections)
Mainly ICU
64% of mortality rates associated to severe nosocomial infections
Recent reports of community acquired infections
Pneumonia (rarely meningitis, cellulitis)
Alcohol abuse, diabetis, cancer, or bronchopulmonary diseases
Possible Vector role
ACINETOBACTER BAUMANNI IS NOW RECOGNIZED AS ONE OF THE KEY MULTI-DRUG RESISTANT ORGANISM REPRESENTING MAJOR CHALLENGES TO HOSPITALS WORLDWIDE
Most infections occur in immunocompromised individuals, and the strain A. baumannii is the second most commonly isolated nonfermenting bacteria in human specimens.
Acinetobacter is frequently isolated in nosocomial infections and is especially prevalent in intensive care units
NEVERTHELESS, RECENTLY REPORTS LINK ACINTOBACTER TO COMMUNITY ACQUIRED INFECTIONS.
Infections by A. baumannii are considered a worrisome phenomenon particularly affecting hospitalized patients, thus being commonly associated with a significantly increase in mortality 5. Wilson S, Knipe C, Zieger M, Gabehart K, Goodman J, Volk H, Sood R. Direct costs of multidrug-resistant Acinetobacter baumannii in the burn unit of a public teaching hospital. Am. J Infect Control. 2004;32:342–344.
Clinical significance
Acinetobacter species are generally considered nonpathogenic to healthy individuals. However, several species persist in hospital environments and cause severe, life-threatening infections in compromised patients.[1] The spectrum of antibiotic resistances of these organisms together with their survival capabilities make them a threat to hospitals as documented by recurring outbreaks both in highly developed countries and elsewhere. An important factor for their pathogenic potential is probably an efficient means of horizontal gene transfer even though such a mechanism has so far only been observed and analyzed in Acinetobacter baylyi, a species that lives in the soil and has never been associated with infections.[1]
Acinetobacter is frequently isolated in nosocomial infections and is especially prevalent in intensive care units, where both sporadic cases as well as epidemic and endemic occurrence is common. A. baumannii is a frequent cause of nosocomial pneumonia, especially of late-onset ventilator associated pneumonia. It can cause various other infections including skin and wound infections, bacteremia, and meningitis, but A. lwoffi is mostly responsible for the latter. A. baumannii can survive on the human skin or dry surfaces for weeks.
Lowman W et al.,J. Med Microbiol. 2008
Garcia-Garmendia JL et al. Clin. Infect. Dis. 2001

5. Genus Acinetobacter 32 described Acinetobacter groups or named species
Gram-negative coccobacillus
Non-fermenters
Oxidase –
Strict aerobes
32 described Acinetobacter groups or named species
Bouvet and Grimont, 1986: Acinetobacter baumannii
A. calcoaceticus- A. baumanii complex (Gerner- Smidt, J. Clin. Microb., 1991)
Acinetobacter baumannii
Acinetobacter calcoaceticus
Genomic species 3TU
Genomic species 13TU
Phenotypically difficult to distinguish
65-75% relatedness in DNA-DNA hybridization
A. baumannii, together with three other closely related Acinetobacter spp., Acinetobacter calcoaceticus and Acinetobacter genomic
species 3 and 13TU, have been grouped together in the ‘A. calcoaceticus–A. baumannii. complex’ because they are very similar
phenotypically and are often impossible to differentiate. A. baumannii and Acinetobacter genomic species 13TU are responsible for most
nosocomial infections, while Acinetobacter genomic species 3 is implicated less often in clinical Disease and A. calcoaceticus is an environmental
organism that has been isolated rarely from clinical specimens.
Micrococcus calcoaceticus – M.W. Beijrinck, 1911
Acinetobacter calcoaceticus
Acinetobacter- 32 species
Outros métodos de identificação: Raman spectrometry, MALDI TOF Mass Spectrometry, recA, e gyrB sequence
Nosocomial infections: A. Baumanni, 13 TU, & 3
Identification: rDNA16S sequence, rpoB sequence, DNA restriction analysis 16S rDNA (ARDRA)
Nosocomial infections: A. baumannii, groups 13TU & 3
Ecological distribution of A. baumannii: undefined reservoir

6. Acinetobacter baumannii - Emergent Pathogen
WHY?
Important factor for their pathogenic potential: efficient means of horizontal gene transfer(?)
Survival capabilities
Acquisition of virulence genes
Ability to acquire resistance to antibiotics
A. baylyi ADP1, A. baumannii AYE and A. baumannii SDF chromosomes. (PLOS One, 2008) ?
baumannii SDF chromosomes was isolated from a human body louse
Multi-drug resistant human clinical isolate strain AYE responsible for a nationwide outbreak in France in 2001

7. An 86-kb Resistance Island in epidemic A. baumannii Strain AYE
Acinetobacter baumannii - Emergent Pathogen Ability to acquire resistance to antibiotics
This study led to the discovery in the AYE genome of an 86-kb region called a resistance “island”—the largest identified to date—that contains a cluster of 45 resistance genes. The homologous location in the susceptible strain, curiously, exhibited a 20-kb genomic island that is devoid of resistance markers. This ability to “switch” its genomic structure probably explains the unmatched speed at which A. baumannii captures resistance markers when under antibacterial pressure, such as is found in hospital intensive care units.
An 86-kb Resistance Island in epidemic A. baumannii Strain AYE
45 resistance genes

8. Acinetobacter baumanii infections- major epidemiological features
Propensity for clonal spread
Involvement in hospital outbreaks
Endemicity in certain hospitals
Resistance to multiple antimicrobial agents
Many recent outbreaks have been caused by multidrug-resistant (MDR) strains of A. baumannii
Similar to methicillin-resistant Staphylococcus aureus, major epidemiological features of these organisms include their propensity for clonal spread, their involvement in hospital outbreaks, and their resistance to multiple antimicrobial agents [2–6]. Many recent outbreaks have been caused by multidrug-resistant (MDR) strains of A. baumannii and have occurred in intensive care units where the extensive use of antibiotics may have contributed to the selection of highly resistant strains [7,8]
Stoeva et al., CMI. 2008
Quinteira et al. AAC. 2004
Manikal et al., CID 2000

9. Acinetobacter isolates resistant to carbapenems
37%
In USA, A. Baumanni is na increasing cause of nosocomial infection in ICU.
City wide outbreak of MDR Ab (42%) resistant to IP.
Asensio et al. Provide useful information about trends in the prevalence of infections caused by carbapenem-resistant A. Baumannnii in Spanish hospitals. Following A DOWNWARD TREND BETWEEN 1997 AND 2002, THEY NOTE THAT CARBAPENEM-RESISTANCE HAS SINCE RISEN AGAIN, REACHING 37% IN 2005.
Most notably, 14.6% of infections were considered communiy-acquired.
Rodriguez-Bano presented a 3.6%...
Perez et al., AAC
Meropenem Yearly Susceptibility Test Information Collection [MYSTIC], 2004.
Asensio et al., Enferm. Infecc. Microb. Clin. 2008
Lolans et al., AAC. 2006

10. Carbapenem resistance in Acinetobacter baumannii
Impaired permeability related to porin changes
Penicillin binding protein modifications
Metallo-β-lactamases (Ambler class B β-lactamases) IMP, VIM, SIM [IMP-5 (one isolate Portugal)]
Carbapenem-hydrolyzing oxacillinases (Ambler class D β-lactamases)
MANY OF THESE NOSOCOMIAL ISOLATES ARE MULTIDRUG RESISTANT INCLUDING TO CARBAPENEMS
SEVERAL MECHANISMS CONTRIBUTE TO CARBAPENEM RESISTANCE, NAMELY IMPERMEABILITY, PBP MODIFICATIONS , METALLO BETA LACTAMASE PRODUCTION AND WITH AN INCREASING RELEVANCE, THE PRODUCTION CARPENEM HYDROLIZING OXACILLINASES .

11. Acquired Carbapenem-Hydrolyzing Oxacillinases
Three diferent groups
OXA-58
<48%
99%
OXA-23 (23,27,49)
60%
99%
OXA-40/24 (25,26,40)
Plasmidic /Chromosomal location

12. Acquired Carbapenem-Hydrolyzing Oxacillinases
Distribution of OXA-23,-25,-26,-27,-40/24,-58
OXA-23 (23,27,49)
OXA-58
OXA-40/24 (25,26,40)
Chromosomal or, mostly, plasmid location
HERE YOU HAVE THE WORLD DISTRIBUTION OF THE ACQUIRED OXACILLINASES
(NICE TALKS ON THIS TOPICS WERE GIVEN BY LAURENT POIREL AND PATRICE NORDMANN IN THE LAST DAYS OF THIS EXCELENT EIGTHEEN ECCMID.)
AS YOU CAN SEE THE MOST DISSEMINATED OXACILLINASE IS OXA FIFTY EIGHT AND OXA TWENTY THREE MOSTLY PLASMID LOCATED
THE OXA FORTY ENZYME WAS FIRST IDENTIFIED IN ACINETOBACTER BAUMANNII ISOLATES FROM IBERIAN PATIENTS AND LATELY IN UNITED STATES.
RECENTLY WE DESCRIBED THE PLASMIDIC LOCATION OF OXA-FORTY BOTH IN ACINETOBACTER BAUMANNII AND IN ACINETOBACTER HAEMOLYTICUS FROM PORTUGAL.
OXA-40 plasmid located in
A. baumannii and A. haemolyticus
Poirel & Nordman, CMI 2006
Lolans et al, AAC 2006
Quinteira et al, AAC 2007
Qi et al, AAC 2008

13. OXA-40 distribution in Portugal1 2 3 4 5 6
1- HPA, Penafiel
Index Case – 1995
OXA-33 (accession no. AY082394) => OXA-40
Da Silva et al., 1999 JCM
2- HGSA, Porto
3- HUC, Coimbra
4- HSM, Lisboa
5- CHCB, Covilhã
6- CHCR, Caldas da Rainha
Da Silva et al., 2004 JAC
IN PORTUGAL, THE FIRST DESCRIPTION OF A CARBAPENEMASE- PRODUCING ACINETOBACTER BAUMANNII WAS IN NINETEEN NINETY FIVE FROM A HOSPITAL ON THE NORTH.
SOON AFTER, AN INCREASING NUMBER OF ISOLATES FROM DIFFERENT HOSPITALS THROUGHOUT OUR COUNTRY WERE PROGRESSIVELY OBSERVED ALL OVER THE LAST YEARS.
ALL ISOLATES WERE CLONALLY RELATED AND OXA FORTY PRODUCERS BELONGING TO THE IBERIAN CLONE
OXA-40 A. baumannii Iberian clone

14. OXA-23 -A. baumannii producers in Portugal
2006
2 isolates; one patient - Hosp. S.Teotónio
37 years old woman with a history of alcohol abuse.
Diagnostic: necrohaemorrhagic pancreatitis
Therapy: meropenem+ fluconazol
Day 9: Imipenem-resistant Ac. baumannii blood and intraperitoneal liquid
Ampicillin and amikacin, given for nineteen days
Isolation of XDR A. baumannii from intraperitoneal liquid
Pancreatic abcess resected
The patient was discharged after 18 weeks of hospitalization.
I-Ceu-I corta no gene rrl
PCR and sequencing revealed bla OXA23
Chromosomal location of bla OXA23
(I-Ceu-I analysis)

15. OXA-23 -A. baumannii producers in Portugal:
Outbreak Hosp. Pedro Hispano (n=8)
Outbreak Hosp., Amarante (n=7)
Hosp. Sto António (n=1)
(bronchial secretions; urine; blood) 5
2007: 1 patient (Community) 2 4 3 1
Subsequentemente bforam encontardos produtores em diferente hospitais todosdomesmo clone, tb disseminado em esopanha=Iberian clone. Um dos hospitais em que a incidencia de resiste aos carabapene os e mto elevada foi o hosp objecto deste estudo
(pus)
PFGE and MLST:
OXA-23 producing clone

16. Conclusions
Emergence of OXA-23 producing A. baumannii in portuguese hospitalised patients
Spread of an OXA-23 producing clone
Associated to outbreaks and sporadic cases
In an ambulatory patient which can further promote community dissemination
With an enlarged resistance profile
An alarming XDR (Extreme Drug Resistant) isolate, that evolved during therapy with ampiciliin and amikacin.
Less resistant strains were already able to disseminate throughout Iberia, causing mortality events.
the inactivity of the antimicrobial agents colistin and tigecycline, recently used as a last therapeutic options for MDR A. baumannii infections.

17. Acknowledgments Francisco Freitas (Hosp. S. Teotónio, Viseu)
Helena Ramos (Centro Hospitalar do Porto, Porto)
Luis Grañeda (Centro Hospitalar Cova da Beira, Covilhã)
Mariana Viana (Hospital de Amarante)
Luísa Cavaleiro e Valquíria Alves (Hospital Pedro Hispano, Matosinhos)
Miguel Monteiro
Filipa Grosso
Sandra Quinteira
Carla Novais
Elisabete Machado
Patrícia Antunes
Ana Raquel Freitas
Raquel Branquinho