Ralstonia mannitolilytica infections in an oncologic day ward: description of a cluster among high-risk patients
Lucarelli et al. Antimicrobial Resistance and Infection Control
Ralstonia mannitolilytica infections in an oncologic day ward: description of a cluster among high-risk patients
Claudia Lucarelli 0 1 3
Enea Gino Di Domenico 2
Luigi Toma 2
Domenico Bracco 2
Grazia Prignano 2
Maria Fortunati 2
Lorella Pelagalli 2
Fabrizio Ensoli 2
Patrizio Pezzotti 1 3
Aurora García-Fernández 1 3
Annalisa Pantosti 1 3
Loredana Ingrosso 0 1 3
0 European Program for Public Health Microbiology Training (EUPHEM), European Centre for Disease Prevention and Control , (ECDC), Stockholm , Sweden
1 Istituto Superiore di Sanità Viale Regina Elena , 299 00161 Rome , Italy
2 Istituto Nazionale Tumori Regina Elena, Istituto Dermatologico San Gallicano , Rome , Italy
3 Istituto Superiore di Sanità Viale Regina Elena , 299 00161 Rome , Italy
Background: Ralstonia spp, an environmental microorganism, has been occasionally associated with healthcare infections. The aim of this study was to investigate an outbreak caused by Ralstonia mannitolilytica in oncology patients. Methods: Case definition: Oncology outpatients attending a day ward, with positive blood and/or central venous catheter (CVC) culture for Ralstonia spp from September 2013 - June 2014. We analysed medical records, procedures and environmental samples. R. mannitolilytica was identified by 16S rRNA sequencing, and typed by Pulsed Field Gel Electrophoresis (PFGE); resistance to carbapenemes was investigated by phenotypic and molecular methods. Results: The patients (N = 22) had different malignancies and received different therapy; all had a CVC and 16 patients presented chills and/or fever. R. mannitolilytica was isolated from both blood and CVC (n = 12) or only blood (n = 6) or CVC tips (n = 4). The isolates had indistinguishable PFGE profile, and showed resistance to carbapenems. All the isolates were negative for carbapenemase genes while phenotypic tests suggests the presence of an AmpC β-lactamase activity,responsible for carbapenem resistance. All patients had had CVC flushed with saline to keep the venous access pervious or before receiving chemotherapy at various times before the onset of symptoms. After the first four cases occurred, the multi-dose saline bottles used for CVC flushing were replaced with single-dose vials; environmental samples were negative for R. mannitolilytica. Conclusions: Although the source of R. mannitolilytica remains unidentified, CVC flushing with contaminated saline solution seems to be the most likely origin of R. mannitolilytica CVC colonization and subsequent infections. In order to prevent similar outbreaks we recommend removal of any CVC that is no longer necessary and the use of single-dose solutions for any parenteral treatment of oncology patients.
Ralstonia mannitolilytica; Outbreak; Central venous catheter; Oncologic patients; Carbapenem resistance
The genus Ralstonia comprises a group of
nonfermentative, Gram-negative bacteria (NFGN) found in
moist environments, such as water, soil and plants .
Three Ralstonia species, Ralstonia pickettii, Ralstonia
insidiosa and Ralstonia mannitolilytica, formerly designated
Burkholderia pickettii, Burkholderia solanacearum and
Pseudomonas thomasii, respectively, have been recognized
as opportunistic human pathogens . Their relevance
has been currently re-evaluated because of their ability
to survive in different types of disinfectants and to pass
through 0.2-μm filters that are used to sterilize
solutions [1, 2].
Ralstonia spp. is reported as a causative agent of
bacteremia [3, 4], meningitis [5, 6], and sepsis [7, 8] in
immunocompromised patients and of central venous
catheter (CVC)-associated bacteremia in oncology
patients [1–4, 9]. Several hospital outbreaks have been
described that were associated with contaminated
solutions, including water for injection, saline solutions,
disinfectants and antiseptics . Multidrug resistance
in NFGN is widely reported in the literature [10, 11]
and is causing increasing concern because such bacteria
may have a role not only as human pathogens but also
as potential reservoirs of resistance genes, particularly
when they are found in hospital settings. Several studies
have described resistance to fluoroquinolones, 3rd
generation cephalosporin and carbapenems  in isolates
belonging to all the three Ralstonia species.
R. pickettii is the Ralstonia species most frequently
reported in the literature while only a limited number
of infections are attributed to R. insidiosa and R.
mannitolilytica . The clinical importance of these two species is
probably underestimated because their biochemical
patterns are similar to that of R. pickettii, making it
impossible their distinction based on conventional
microbiological tests only [1, 4, 12, 13].
Here we describe an outbreak caused by R.
mannitolilytica in patients attending a day ward unit in an
oncology hospital in Rome occurred from September 2013 –
The Istituto Nazionale Tumori Regina Elena – Istituto
Dermatologico San Gallicano is a 215-bed hospital
located in Rome with approximately 6,700 inpatient
admissions and 900,000 outpatients visits per year. The
hospital has two oncology day wards (DW-A and DW-B)
located in different buildings, each consisting of a single
therapy room with 20 bays for chemotherapy infusion and
attended by an average of 65 patients/day. Patients are
assigned to either ward while the infusion bays are
not pre-assigned neither a register for the allocation
of patients to bays is in place. One of the units (DW-A)
was affected by the outbreak and was the object of the
epidemiological investigation. From September 2013 – June
2014, 2485 patients attended DW-A for a total of 20,177
We defined a case as a patient attending DW-A from
September 2013 – June 2014 that had a blood culture
and/or a CVC tip culture positive for Ralstonia spp. with
or without symptoms (chills and/or fever). Two of the
authors (CL, LI) assisted by an infection control nurse
(MF) reviewed all the medical records of the cases to
identify any common medical procedure or any occurrence
that might have posed a risk for acquiring Ralstonia spp.
Environmental sampling started on 17 October 2013,
immediately after Ralstonia spp. had been isolated from
the blood culture of the third case. Laboratory
technicians collected the samples using commercially available
sterile swabs (COPAN Eswabs, Brescia, Italy), following
existing departmental guidelines, in the therapy room
from furniture and electronic devices (N = 8), and in the
drug preparation room (N = 8) from personal computer,
telephone, fax, medicine cabinet, medicine trolley. Swabs
were cultured in Tryptic Soy Broth, incubated for 48 h
at 37°C, and plated on chocolate agar and blood agar.
Samples were also obtained from liquid soaps (N = 10),
their dispensers (N = 8) and chlorhexidine (N = 4) in use
in DW-A. In addition distilled water and sterile water
used for injection were cultured as described in Moreira
et al. .
Microbiological methods and typing of isolates
Blood samples were taken through CVC in all patients. R.
mannitolilytica was isolated from signal-positive blood
culture bottles (BacT/ALERT BioMérieux, Florence, Italy)
using conventional methods. Sixteen CVC tips were
cultured according to Zhang et al. .
Identification of the isolates was obtained by VITEK 2
system (BioMérieux, Florence, Italy). Identification at the
species level was obtained by amplification of the 16S
rDNA gene followed by double-strand sequencing .
To investigate the relatedness of Ralstonia isolates,
PFGE was performed on all isolates obtained from blood
cultures and CVC tip cultures, following digestion of
genomic DNA by the restriction enzyme SpeI (New Englands
Biolabs, Ipswich, MA), according to the CDC protocol
https://www.cdc.gov/pulsenet/pdf/ecolishigella-salmonella-pfge-protocol-508c.pdf ) with
addition of 50 μM thiourea in the agarose gel and in
the electrophoresis running buffer. The following
running conditions were used: 120 costant angle at 6V/
cm, with pulse time 20h 1s-40s, 4h 30s-60s. Salmonella
Braenderup H9812 was used as reference for molecular
All the experiments were performed using two controls
strains: R. mannitolilytica BK931  and R. mannitolilytica
ATCC BAA-716 (LMG 6866) .
Antibiotic susceptibility of the isolates
Susceptibility testing of the isolates was performed by the
disk diffusion method according to the EUCAST guidelines
(available at http://www.eucast.org/clinical_breakpoints/).
The antimicrobials tested were: ceftazidime, meropenem,
ciprofloxacin, gentamicin, amikacin, and piperacillin/
tazobactam (Becton Dickinson, Milan, Italy).
Antimicrobial susceptibility to piperacillin/tazobactam was
confirmed by Etest (BioMérieux, Florence, Italy). As there are
no EUCAST susceptibility breakpoints available for
Ralstonia spp., the results were interpreted using the
EUCAST criteria for Pseudomonas spp. Pseudomonas
aeruginosa ATCC 27853 was included as control.
Identification of determinants of carbapenem-resistance
Four outbreak isolates (A, E, L, S) were randomly selected
to perform the phenotypic and molecular tests.
Identification of resistance mechanisms for carbapenems was
performed by the agar tablet/disc diffusion method (KPC/
MBL and OXA-48 Confirm Kit, ROSCO Diagnostica A/S,
Taastrup, Denmark). In addition, PCR assays were
performed for the identification of the chromosomal genes
blaOXA-443 and blaOXA-444, previously described in a
carbapenem-resistant R. mannitolilytica strain . For
amplification of the two genes, two couple of primers
were designed: OXA-443 Fw 5’-ATGACGAAACTCC
GCCA-3’/OXA-443 Rv 5’-AGGTGGGCTCGATCTTG-3’
and OXA-444 Fw 5’-ATGTTCTCTCGTTGGTC-3’/
OXA-444 Rv 5’- TGCGGGTCGGACGGAGA -3’. The
presence of other carbapenem-resistance genes was
investigated by multiplex PCR assay with primers designed to
amplify the following 11 genes: blaIMP, blaVIM, blaNDM,
blaSPM, blaAIM, blaDIM, blaGIM, blaSIM, blaKPC, blaBIC, and
blaOXA-48, accordingly to Poirel et al. .
Epidemiological and microbiological investigation
According to case definition, we identified 22 patients
(attack rate 0,88%), 13 males and 9 females, age range
30–84 years old (median age 66), attending DW-A from
24 September 2013 – 23 June 2014 (Fig. 1, Table 1). In
particular, 12 patients had Ralstonia spp. positive
cultures from both blood and the CVC tip; four patients
didn’t have blood culture performed, but because of the
clinical symptoms their catheters were removed and
cultured and were found positive for Ralstonia spp.; other
six patients had a positive blood culture but the CVC
tips were not tested.
Analysis of the medical records revealed that the
patients had different types of CVC, in particular 20 had a
Port, while two had a PICC (percutaneous introduction
central catheter). We were able to retrieve the insertion
dates of the devices for 13 out of 22 patients: they were
inserted from more than 4 years to less than 2 months
before the onset of symptoms (Table 1). The patients
had different types of solid cancer, they underwent
different therapeutic protocols and attended DW-A in
different days. The only common procedure among the
patients that we were able to identify was a maintenance
procedure, which consisted in flushing saline solution
through the CVC, after disinfection with 2% chlorhexidine
gluconate in 70% isopropyl alcohol. Such procedure was
performed either before the patient received
chemotherapy or as a periodic standard procedure to keep the
patient’s venous access pervious. Sixteen patients reported
fever and/or chills while receiving chemotherapy through
CVC or within two hours after the CVC flushing
procedure. No information was available for the remaining six
patients. No patient had signs of skin infection at the Port
Fig. 1 Epidemic curve of cases of R. mannitolilytica in oncology patients attending the day hospital ward A, September 2013-June 2014
Table 1 Data of the 22 cases (A-V) of R. mannitolilytica outbreak, September 2013-June 2014, oncology hospital in Rome
NA: not available; ND: not determined
site or at the PICC insertion site. Empirical antibiotic
therapy (ciprofloxacin) was deemed necessary for one patient
only. All patients had their CVCs removed and this led to
resolution of symptoms in all cases.
The review of the current medical practices of
DWA revealed that, between August 2013 and September
2013, five bottles of saline solution of 250 ml were
used for CVC flushing. This type of bottle was in use
only in DW-A, and not in DW-B, and usually
remained in use for two days from opening. All the
cases attended the DW-A for CVC flushing when the
250 ml bottles of saline solution were in use. After
the occurrence of the first three cases, the hospital
committee for control of healthcare-associated
infection gave indications to perform environmental
cultures and room disinfection (mid October). However,
since the 250 ml bottles of saline solution had been
already discarded and replaced with single-dose vials,
they were unavailable for microbiological testing when
the investigation was started. All the environmental
samples and samples from chlorhexidine, distilled
water and sterile water were negative for the presence
of Ralstonia spp.
Characterization of the pathogen
Ralstonia spp. had never been isolated from any patients
in the hospital both before and after the outbreak. By
routine biochemical tests the isolates from blood and
CVC tips were identified as R. pickettii. However, 16S
rDNA sequencing showed that the isolates were in fact
R. mannitolilytica. In addition, all the isolates shared an
indistinguishable PFGE profile (Fig. 2) while the two
control strains (ATCC BAA-716 and BK931), showed
profiles that were distinct from those of the outbreak
strains. The outbreak strains had the same multi-drug
resistance profile: they were resistant to ceftazidime,
meropenem, ciprofloxacin, gentamicin, and amikacin but
were susceptible to piperacillin/tazobactam (MIC ≤16
mg/L). Except for susceptibility to ciprofloxacin, the two
control strains had the same resistance pattern.
Regarding resistance to carbapenems the strains were
negative for the presence of the 11 carbapenemase genes
screened by multiplex PCR. All the isolates tested,
including the two controls, were positive by PCR for the
blaOXA443 and blaOXA-444 genes. Phenotypic tests performed to
assess the mechanism of carbapenem resistance showed
that cloxacillin was able to abolish meropenem resistance,
Fig. 2 Pulsed-field gel electrophoresis (PFGE) of R. mannitolilytica
strains. M: Molecular weight standard, Salmonella Braenderup H9812.
A: strain isolated from blood culture. U, V: strains isolated from CVC.
These strains are representative of the entire sample. BK931, ATCC
BAA-716: control strains
The microbiological and epidemiological investigation of
this outbreak did not detect the source of the
contamination, however the molecular typing of the pathogen
strongly supports the hypothesis of a common source of
contamination. Failure to identify the culprit is probably
due to the fact that when the investigation was started
samples from disinfectants, antiseptics and saline
solutions used at the beginning of the outbreak were not
available for microbiological investigation. In fact,
disinfectants, antiseptics and multi-dose bottles of saline
solution have been described in the literature as one of the
main sources for Ralstonia spp. contamination ,
therefore their prompt removal from DW-A
immediately after the beginning of the outbreak was the most
understandable precaution to adopt from the patients’
safety viewpoint. However, in spite of the multi-dose
bottles of saline solution removal, the outbreak lasted
9 months. Indeed, the revision of the medical records
showed that CVC flushing with saline solution was a
common procedure adopted with all the patients and that, at
the beginning of the outbreak, multi-dose bottles of saline
solution were in use in DW-A. Thus it is very likely that
patients were exposed to one or more contaminated
bottle(s) of saline. The majority of the patient developed
clinical symptoms immediately thereafter, while others likely
had a CVC colonization by R. mannitolilytica. Any
subsequent procedure through CVC, chemotherapy or new
saline flushing, may have caused detachment and
dissemination of R. mannitolilytica from CVC, causing fever and
chills. This might have happened even after several weeks
from CVC colonization, possibly accounting for the
protracted duration of the outbreak. A similar finding have
been reported by Raveh et al.  in which, a patient
with a CVC developed fever and chills and his blood
culture was positive for R. pickettii. The patient was
treated with antibiotic therapy and the CVC was left in
place. However 15 months later the patient developed
another bloodstream infection with the same organism
likely colonizing CVC.
The ability to grow as biofilm on abiotic surfaces
plays an important role in colonization of hospital
equipment and indwelling medical devices such as
CVCs . Ability to form biofilm has been
described in different Ralstonia species including also
R. mannitolylitica [21–24]. It is conceivable that biofilm
formation might have played a role for these strains to
allow adherence to CVCs and subsequent dissemination
in the host, following the flushing procedures of the
The prolonged duration of this outbreak is not
unprecedented for this pathogen. Daxboeck et al.  reported
isolation of R. mannitolilytica in 30 patients attending
15 different wards between February 2002 and March
2004; in this study also the source was not identified.
Other outbreaks have been reported in pediatric
patients, associated with oxygen delivery devices [25, 26]
and in oncology patients [1, 2]. In the outbreak
described by Gröbner et al. , which lasted 11 weeks,
the source was not identified but it was hypothesized
that a contaminated solution administered through
CVC could have been the culprit.
It is important to underline that the misuse of multidose
vials, as in our outbreak, has been frequently reported as a
cause of numerous outbreaks [27–30] in different
European countries as well as in USA where the CDC is aware
of at least 49 outbreaks occurred in 13 years .
Therefore the correct use of single dose vials, for a single
patient, is an effective and important way to prevent
outbreaks as reported by specific guidelines .
R. mannitolilytica is frequently recognized as a
multiresistant microorganism [4, 33–35] but limited data are
available about carbapenem-resistance. To the best of our
knowledge, only a limited number of studies so far, have
tested susceptibility to this drug: in four studies, all the
strains tested were resistant (five in one study and one
strain each in the other three) [2, 17, 33] while Daxboeck
et al.  reported carbapenem-resistance in 12 strains out
of 30. In our isolates no carbapenemase genes were
detected, but they were positive for the chromosomal genes
blaOXA-443 and blaOXA-444, that have been previously
described in one R. mannitolilytica carbapenem-resistant
strain  and that bear close similarity to the blaOXA-22
and blaOXA-60 genes found in R. pickettii . blaOXA-22
and blaOXA-60 code for two oxacillinase, a narrow
spectrum oxacillinase and an inducible carbapenemase,
respectively, although none of them was found to be
responsible for carbapenem resistance in R. pickettii [36,
37]. In addition, the cloxacillin test suggested that the
R. mannitolilytica isolates were positive for the
presence of AmpC β-lactamases. These enzymes are
clinically important because they may confer resistance to a
wide variety of β-lactam drugs, narrow-, expanded-,
and broad-spectrum cephalosporins,
β-lactam-β-lactamase-inhibitor drugs combinations, as well as to
aztreonam and carbapenems in case of association with
altered porins and/ or efflux mechanisms . Taken
together, our data suggest that meropenem resistance
was likely due to overproduction of AmpC β-lactamase,
possibly in synergy with a second mechanism (e.g.
decreased production of the porin channel and/or
activation of efflux systems). Recently, a serin-hydrolase class
C family β-lactamase has been identified in R.
mannitolilytica (GenBank accession number: WP_045219476)
, therefore production of this enzyme may
contribute to carbapenem resistance.
Although R. mannitolilytica did not cause
lifethreatening infections in this outbreak as well as in
previously reported outbreaks [2, 26], the increased
detection of Ralstonia spp. in healthcare settings coupled
with the emergence of multi-resistant strains of R.
mannitolilytica, represent a reason of concern,
particularly in case of vulnerable patients which may require
an antimicrobial therapy.
We reported the first outbreak due to R. mannitolilytica
in oncology patients bearing CVC in Italy. Although the
source of the outbreak could not be readily identified,
the investigation suggested that contaminated saline
solution used for CVC flushing may have been the source
of the outbreak. In order to prevent possible infections
we recommend the removal of any CVC that is not
longer necessary and the use of single dose solutions for
any parenteral treatment of cancer patients.
CVC: Central venous catheter; DW-A: Day ward A; DW-B: Day ward B;
MIC: Minimal inhibitory concentration; NFGN: Non-fermentative
gramnegative bacteria; PCR: Polymerase chain reaction; PFGE: Pulsed field gel
electrophoresis; PICC: Percutaneous introduction central catheter
Availability of data and materials
Please contact author for any requests.
CL performed PFGE, antimicrobial susceptibility test and initial draft
preparation. LT, DB, GP, EGDD, MF, LP, FE CL, AP, LI, PP, participated in
the outbreak investigation. AGF performed carbapenem resistance
characterization. LI, AP, PP, EGDD, FECLfinalized the manuscript. All
authors read and approved the final manuscript.
The authors declare that they have no competing interests.
Consent for publication
1. Ryan MP , Adley CC . Ralstonia spp.: emerging global opportunistic pathogens . Eur J Clin Microbiol Infect Dis . 2014 ; 33 ( 3 ): 291 - 304 .
2. Gröbner S , Heeg P , Autenrieth IB , Schulte B. Monoclonal outbreak of catheter-related bacteraemia by Ralstonia mannitolilytica on two haematooncology wards . J Infect . 2007 ; 55 ( 6 ): 539 - 44 .
3. Mikulska M , Durando P , Molinari MP , Alberti M , Del Bono V , Dominietto A , et al. Outbreak of Ralstonia pickettii bacteraemia in patients with haematological malignancies and haematopoietic stem cell transplant recipients . J Hosp Infect . 2009 ; 72 ( 2 ): 187 - 8 .
4. Daxboeck F , Stadler M , Assadian O , Marko E , Hirschl AM , Koller W. Characterization of clinically isolated Ralstonia mannitolilytica strains using random amplification of polymorphic DNA (RAPD) typing and antimicrobial sensitivity, and comparison of the classification efficacy of phenotypic and genotypic assays . J Med Microbiol . 2005 ; 54 (Pt 1): 55 - 61 .
5. Ann HM . An unusual case of bacterial meningitis caused by Burkholderia pickettii . Clin Microbiol Newsl . 1998 ; 102 - 3 .
6. Bonatti H , Stelzmueller I , Laimer I , Obwegeser A. Ralstonia pickettii meningitis in a child with hydrocephalus . Eur J Pediatr Surg . 2009 ; 19 ( 5 ): 341 - 2 .
7. Marroni M , Pasticci MB , Pantosti A , Colozza MA , Stagni G , Tonato M. Outbreak of infusion-related septicemia by Ralstonia pickettii in the Oncology Department . Tumori. 2003 ; 89 ( 5 ): 575 - 6 .
8. Weist K , Stolze H , Sohr D , Wickmann L , Liebeskind AK , Rüden H , et al. P14 . 03 Ralstonia pickettii septicemia in pediatric oncology patients associated with the use of contaminated heparin-saline-solution . J Hosp Infect . 2006 ; 64 (Supplement 1): S74 .
9. Pasticci MB , Baldelli F , Camilli R , Cardinali G , Colozza A , Marroni M , et al. Pulsed field gel electrophoresis and random amplified polymorphic DNA molecular characterization of Ralstonia pickettii isolates from patients with nosocomial central venous catheter related bacteremia . New Microbiol . 2005 ; 28 ( 2 ): 145 - 9 .
10. Enoch DA , Birkett CI , Ludlam HA . Non-fermentative Gram-negative bacteria . Int J Antimicrob Agents . 2007 ;29 Suppl 3: S33 - 41 .
11. McGowan JE . Resistance in nonfermenting gram-negative bacteria: multidrug resistance to the maximum . Am J Med . 2006 ; 119 ( 6 Suppl 1 ): S29 - 36 . discussion S62- 70 .
12. Ryan MP , Pembroke JT , Adley CC . Differentiating the growing nosocomial infectious threats Ralstonia pickettii and Ralstonia insidiosa . Eur J Clin Microbiol Infect Dis . 2011 ; 30 ( 10 ): 1245 - 7 .
13. De Baere T , Steyaert S , Wauters G , Des Vos P , Goris J , Coenye T , et al. Classification of Ralstonia pickettii biovar 3/'thomasii' strains (Pickett 1994 ) and of new isolates related to nosocomial recurrent meningitis as Ralstonia mannitolytica sp . nov. Int J Syst Evol Microbiol . 2001 ; 51 (Pt 2): 547 - 58 .
14. Moreira BM , Leobons MB , Pellegrino FL , Santos M , Teixeira LM , de Andrade ME , Sampaio JL , Pessoa-Silva CL . Ralstonia pickettii and Burkholderia cepacia complex bloodstream infections related to infusion of contaminated water for injection . J Hosp Infect . 2005 ; 60 ( 1 ): 51 - 5 .
15. Zhang L , Morrison M , Rickard CM . Draft Genome Sequence of Ralstonia pickettii AU12-08, Isolated from an Intravascular Catheter in Australia . Genome Announc . 2014 ; 2 ( 1 ): e0027 - 14 .
16. Eilmus S , Heil M. Bacterial associates of arboreal ants and their putative functions in an obligate ant-plant mutualism . Appl Environ Microbiol . 2009 ; 75 ( 13 ): 4324 - 32 .
17. Suzuki M , Nishio H , Asagoe K , Kida K , Suzuki S , Matsui M , et al. Genome Sequence of a Carbapenem-Resistant Strain of Ralstonia mannitolilytica . Genome Announc . 2015 : 3 ( 3 ) e00405 - 15 .
18. Poirel L , Walsh TR , Cuvillier V , Nordmann P. Multiplex PCR for detection of acquired carbapenemase genes . Diagn Microbiol Infect Dis . 2011 ; 70 ( 1 ): 119 - 23 .
19. Raveh D , Simhon A , Gimmon Z , Sacks T , Shapiro M. Infections caused by Pseudomonas pickettii in association with permanent indwelling intravenous devices: four cases and a review . Clin Infect Dis . 1993 ; 17 ( 5 ): 877 - 80 .
20. Donlan RM . Biofilms and device-associated infections . Emerg Infect Dis . 2001 ; 7 ( 2 ): 277 - 81 .
21. Hu JY , Fan Y , Lin YH , Zhang HB , Ong SL , Dong N , et al. Microbial diversity and prevalence of virulent pathogens in biofilms developed in a water reclamation system . Res Microbiol . 2003 ; 154 ( 9 ): 623 - 9 .
22. Kang Y , Liu H , Genin S , Schell MA , Denny TP . Ralstonia solanacearum requires type 4 pili to adhere to multiple surfaces and for natural transformation and virulence . Mol Microbiol . 2002 ; 46 ( 2 ): 427 - 37 .
23. Zhang L , Gowardman J , Morrison M , Krause L , Playford EG , Rickard CM . Molecular investigation of bacterial communities on intravascular catheters: no longer just Staphylococcus . Eur J Clin Microbiol Infect Dis . 2014 ; 33 ( 7 ): 1189 - 98 .
24. Di Domenico EG , Toma L , Christian C , Ascenzioni F , Sperduti I , Prignano G , et al. Development of an in vitro Assay, Based on the BioFilm Ring Test®, for Rapid Profiling of Biofilm-Growing Bacteria . Front Microbiol . 2016 ; 7 : 1429 . Published online 2016 Sep 21 . doi: 10.3389/fmicb.2016.01429.
25. Block C , Ergaz-Shaltiel Z , Valinsky L , Temper V , Hidalgo-Grass C , Minster N , et al. Déjà vu: Ralstonia mannitolilytica infection associated with a humidifying respiratory therapy device , Israel, June to July 2011 . Euro Surveill . 2013 ; 18 ( 18 ): 20471 .
26. Jhung MA , Sunenshine RH , Noble-Wang J , Coffin SE , St John K , Lewis FM , et al. A national outbreak of Ralstonia mannitolilytica associated with use of a contaminated oxygen-delivery device among pediatric patients . Pediatrics . 2007 ; 119 ( 6 ): 1061 - 8 .
27. Pan A , Dolcetti L , Barosi C , Catenazzi P , Ceruti T , Ferrari L , Magri S , et al. An outbreak of Serratia marcescens bloodstream infections associated with misuse of drug vials in a surgical ward . Infect Control Hosp Epidemiol . 2006 ; 27 ( 1 ): 79 - 82 .
28. Nakashima AK , Highsmith AK , Martone WJ . Survival of Serratia marcescens in benzalkonium chloride and in multiple-dose medication vials: relationship to epidemic septic arthritis . J Clin Microbiol . 1987 ; 25 ( 6 ): 1019 - 21 .
29. Harbarth S , Sudre P , Dharan S , Cadenas M , Pittet D. Outbreak of Enterobacter cloacae related to understaffing, overcrowding, and poor hygiene practices . Infect Control Hosp Epidemiol . 1999 ; 20 ( 9 ): 598 - 603 .
30. Muller AE , Huisman I , Roos PJ , Rietveld AP , Klein J , Harbers JB , Dorresteijn JJ , et al. Outbreak of severe sepsis due to contaminated propofol: lessons to learn . J Hosp Infect . 2010 ; 76 ( 3 ): 225 - 30 .
31. Preventing infection from the misuse of vials . The Joint Commission Issue 52 , June 16, 2014 https://www.jointcommission.org/assets/1/6/SEA_52.pdf. Accessed 9 Jan 2017 .
32. Protect Patients Against Preventable Harm from Improper Use of SingleDose/Single-Use Vials . https://www.cdc.gov/injectionsafety/cdcpositionsingleusevial.html. Accessed 9 Jan 2017 .
33. Dotis J , Printza N , Orfanou A , Papathanasiou E , Papachristou F. Peritonitis due to Ralstonia mannitolilytica in a pediatric peritoneal dialysis patient . New Microbiol . 2012 ; 35 ( 4 ): 503 - 6 .
34. Maroye P , Doermann HP , Rogues AM , Gachie JP , Mégraud F. Investigation of an outbreak of Ralstonia pickettii in a paediatric hospital by RAPD . J Hosp Infect . 2000 ; 44 ( 4 ): 267 - 72 .
35. Vaneechoutte M , De Baere T , Wauters G , Steyaert S , Claeys G , Vogelaers D , et al. One case each of recurrent meningitis and hemoperitoneum infection with Ralstonia mannitolilytica . J Clin Microbiol . 2001 ; 39 ( 12 ): 4588 - 90 .
36. Nordmann P , Poirel L , Kubina M , Casetta A , Naas T. Biochemical-genetic characterization and distribution of OXA-22, a chromosomal and inducible class D beta-lactamase from Ralstonia (Pseudomonas) pickettii . Antimicrob Agents Chemother . 2000 ; 44 ( 8 ): 2201 - 4 .
37. Girlich D , Naas T , Nordmann P. OXA-60, a chromosomal, inducible, and imipenem-hydrolyzing class D beta-lactamase from Ralstonia pickettii . Antimicrob Agents Chemother . 2004 ; 48 ( 11 ): 4217 - 25 .
38. Wozniak A , Villagra NA , Undabarrena A , Gallardo N , Keller N , Moraga M , et al. Porin alterations present in non-carbapenemase-producing Enterobacteriaceae with high and intermediate levels of carbapenem resistance in Chile . J Med Microbiol . 2012 ; 61 (Pt 9): 1270 - 9 .