FEMS Microbiology Letters 228 (2003) 175^179
www.fems-microbiology.org
Antimicrobial resistance in Gram-negative bacilli isolated from
infant formulas
Let|¤cia A.M. Carneiro, Ana P.S. Silva, Va“nia L.C. Merquior, Mara L.P. Queiroz
Disciplina de Microbiologia e Imunologia, Faculdade de Cie“ncias Me¤dicas, Universidade do Estado do Rio de Janeiro, Avenida 28 de Setembro 87, Fundos,
3‡ andar, 20551-030 Rio de Janeiro, RJ - Brazil
Received 16 July 2003; received in revised form 12 August 2003; accepted 22 September 2003
First published online 14 October 2003
Abstract
A total of 90 samples of infant formula (IF) were collected from the lactary of a teaching hospital, during a 4-month period from July
to August 1999. The sanitary conditions of the formulas were analyzed, and a physiological characterization of Gram-negative bacillus
isolates and antimicrobial susceptibility testing were performed. Colony counts were considered to be unacceptable for the majority of the
IF samples and the contamination rates were related to inadequate handling. Coliforms (35‡C and 45‡C growth) were detected in most of
the IF tested. Klebsiella pneumoniae, Citrobacter freundii, Cedacea davisae, Klebsiella planticola and Enterobacter cloacae were the isolates
most commonly identified. Antimicrobial susceptibility testing showed significant resistance rates, particularly to amoxicillin/clavulanic
acid, cefoxitin, cephalotin or ampicillin. One extended-spectrum L-lactamase-producing K. pneumoniae strain was also recovered.
< 2003 Federation of European Microbiological Societies. Published by Elsevier B.V. All rights reserved.
Keywords : Infant formula ; Gram-negative bacillus; Antimicrobial resistance
1. Introduction
Newborns that cannot be fed naturally and require nutritional support, particularly those with low birth weight,
are often nutritionally and immunologically compromised
[1]. Immunosuppression, stress, administration of antibiotics or antacids play an important role in altering defense
mechanisms, allowing microorganisms present in food to
replace the normal gastrointestinal £ora. The importance
of the colonization of skin and mucosa has been recognized as a crucial step in the development of nosocomial
infections [2]. Additionally, the intestinal environment is a
scenario of selective processes due to therapeutic and prophylactic use of antimicrobial agents representing an imperceptible reservoir of multiresistant bacteria [3].
Contamination of nutritional formulas has been implicated in the etiology of nosocomial infections, especially
when administered to immunocompromised patients. The
association with gastrointestinal symptoms or other infec-
* Corresponding author. Tel. : +55 (21) 2587-6380;
Fax : +55 (21) 2587-6476.
E-mail address : [email protected] (M.L.P. Queiroz).
tious consequences such as bacteremia has also been demonstrated [1,2,4,5]. There is evidence showing phenotypic
and genotypic similarities between isolates from human
infections and those recovered from food and nutritional
formulas given to hospitalized patients [2,4,5]. Studies conducted in England, Belgium and Spain have demonstrated
that bacterial strains with indistinct plasmid pro¢les and
antimicrobial susceptibility patterns were isolated from enteral formulas and from blood of patients who had received the formula [2,4,5]. Furthermore, a statistical association was found between the intestinal colonization of
hospitalized children with multiresistant bacteria and arti¢cial milk feedings, antimicrobial use, low weight and long
periods of hospitalization [6].
The contamination of infant formulas (IF) can occur
during preparation, especially when modi¢cation, supplementation and/or reconstitution procedures are required,
or during their storage and transportation [1,7]. Nosocomial outbreaks as a result of IF contamination have been
reported worldwide with special reference to Gram-negative bacilli such as Klebsiella spp., Enterobacter spp., Escherichia coli, Pseudomonas aeruginosa, Serratia marcescens and Salmonella spp. [1,2,4,5]. Considering the high
rates of mortality and the extensive usage of antimicrobial
agents in neonatal intensive care units (NICU), the aim of
0378-1097 / 03 / $22.00 < 2003 Federation of European Microbiological Societies. Published by Elsevier B.V. All rights reserved.
doi:10.1016/S0378-1097(03)00739-0
FEMSLE 11247 12-11-03
176
L.A.M. Carneiro et al. / FEMS Microbiology Letters 228 (2003) 175^179
this study was to determine the sanitary conditions as well
as the presence and antimicrobial susceptibility pro¢les of
Gram-negative bacilli isolated from IF that were prepared
at the lactary of a teaching hospital located in Rio de
Janeiro, Brazil.
with colony counts higher than 102 CFU ml31 to be unacceptable, as described by others [1,11].
Up to ¢ve di¡erent colony types of organism grown
from each sample were identi¢ed as a species in Enterobacteriaceae and suspected colonies on King B agar were
identi¢ed as P. aeruginosa using suitable biochemical tests
described elsewhere [12].
2. Materials and methods
2.3. Antimicrobial susceptibility testing
2.1. Milk samples
A total of 90 samples of IF (o¡ered to low birth weight
neonates) were collected during a 15-week period (from
July to November, 1999) from the lactary of the Hospital
Universita¤rio Pedro Ernesto (HUPE/UERJ), a tertiarycare teaching hospital located in the city of Rio de Janeiro,
Brazil. Regarding the procedures adopted by the institution analyzed, the total volume of the formula destined for
the infants is prepared in the late morning, poured into
plastic bottles according to the number of single-dose feedings that each infant should receive during the day and
maintained at 4‡C. The formula is prepared by two groups
of dietary technicians working on alternate days. Thus, the
IF samples (40 ml each) were collected during di¡erent
shifts. The two groups had the same training background
and adopted the same procedures in the preparation of the
formula. Moreover, the time between its preparation and
distribution was determined and each of the 15 samples
prepared by each one of the groups were tested immediately after preparation and after 6 and 20 h of refrigeration, making up a total of 45 analyses per group of
technicians. Milk samples were collected into sterile
containers, transported under refrigeration and immediately processed at the laboratory.
2.2. Sample analysis
Quantitative analysis was performed and the presence of
Enterobacteriaceae and P. aeruginosa was investigated.
The quantitative microbiological analysis of the IF included aerobic plate colony counts at 35‡C, determination
of the most probable number (MPN) of coliform organisms at 35‡C and 45‡C and enumeration of coagulase-positive staphylococci as done by American Public Health
Association [8]. Enterobacteriaceae strains were isolated
by methods described previously [8].
Additionally, the presence of P. aeruginosa was evaluated by plating 0.1 ml of the samples on to King B
agar [9].
The results were evaluated according to the following
standards limits established by the Brazilian legislation
[10]: absence of fecal coliforms (at 45‡C), Salmonella
and coagulase-positive staphylococci. Moreover, IF samples may contain up to 10 MPN of coliform organisms at
35‡C (total coliforms). Additionally, since there is no standard for colony counts for IF samples, we de¢ned samples
Isolates were tested by disk di¡usion according to the
recommendations of the National Committee for Clinical
Laboratory Standards [13]. The following antimicrobial
drugs were tested: amikacin, cefotaxime, ceftazidime,
chloramphenicol, gentamicin, imipenem, netilmicin, tetracycline and sulfamethoxazole/trimethoprim for all organisms. Enterobacteriaceae strains were also tested against
amoxicillin-clavulanic acid, ampicillin, cefoxitin and cephalotin. Likewise, cipro£oxacin, piperacillin, ticarcillin-clavulanic acid, and tobramycin were included as additional
tests against P. aeruginosa. E. coli ATCC25922, E. coli
ATCC35218, P. aeruginosa ATCC27853 and Staphylococcus aureus ATCC25923 were used as control strains. Double-disk di¡usion assays with ceftazidime and ceftazidime+clavulanic acid and cefotaxime and cefotaxime+
clavulanic acid were performed for the detection of extended-spectrum L-lactamases (ESBL) among strains
showing resistance to any third generation cephalosporin.
Reference strain E. coli ATCC25922 was used as control
strain.
2.4. Statistical analysis
The M2 test performed with the Epi-Info statistical program (version 6.0; Centers for Diseases Control and Prevention, Atlanta, GA, USA) was used to compare the
di¡erences between the two groups of dietetic technicians
with number of contaminated IF samples at each time of
storage. Bonferroni tests (GraphPad Prism, version 3.0,
GraphPad Software, San Diego, CA, USA) were used to
perform variance analysis of quantitative results with each
period of IF storage. Statistical signi¢cance was inferred
for comparisons when P 6 0.05.
3. Results
The sanitary conditions of the IF varied according to
the group of dietetic technicians that worked in their preparation. Coliform counts higher than 10 MPN at 35‡C
were not observed among samples prepared by group B,
while 29 (64.4%) of the IF samples prepared by group A
showed unacceptable counts (P 6 0.05). In the same manner, the number of samples presenting coliform contamination at 45‡C was eight-fold higher among the samples
prepared by group A (35.5%) than among those prepared
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177
Table 1
Percentage of unacceptable IF samples regarding the period of storage according to the standards set by Brazilian legislationa
0 hb (n = 15)
A
Colony counts
Coliform growth at 35‡C
Coliform growth at 45‡C
c
6 h (n = 15)
c
20 h (n = 15)
B
A
B
A
B
13.3
0
0
73.3
73.3
46.6
33.3
0
6.6
68.8
66.6
33.3
24.4
0
6.6
60.0
53.3
26.6
a
9 102 CFU ml31 ; 9 10 MPN of total coliforms and absence of fecal coliforms.
Tested immediately after the preparation.
c
Groups of dietary technicians that worked on the preparation of the formulas.
b
resistance to at least one antimicrobial agent tested. Moreover, resistance or intermediate resistance to 11 antimicrobial agents was observed. Among the Enterobacteriaceae,
55 (33.3%) strains isolated from 18 IF samples were resistant or showed intermediate resistance to the same combination of four antimicrobial agents: ampicillin, amoxicillin/clavulanic acid, cefoxitin and cephalotin. Resistance to
at least one antimicrobial agent within this group of four
drugs was observed in all strains. Although all strains were
susceptible to cefotaxime, netilmicin and imipenem, the
K. pneumoniae strain that was resistant to ceftazidime
was found be an ESBL producer. Eight other K. pneumoniae strains were resistant to aminoglycosides. All P. aeruginosa isolates were resistant to chloramphenicol and tetracycline and were susceptible to imipenem and
ceftazidime. The resistance patterns of isolates are summarized in Table 3.
by group B (4.4%) (P 6 0.05). From the total of IF samples prepared by groups A and B, 31 (68.8%) and 11
(24.4%) presented colony counts above 2U103 CFU
ml31 , respectively (P 6 0.05). The presence of coagulasepositive staphylococci was not observed in any IF sample.
The proportion of IF samples unacceptable according
to Brazilian legislation [10] increased after storage periods
(Table 1). Among the samples tested immediately after
their preparation, nine (60%) prepared by group A and
two (13.3%) by group B demonstrated unacceptable colony counts. After 6 h of storage these numbers reached 11
(73.3%) and ¢ve (33.3%), respectively. In the same manner, the number of samples prepared by group A contaminated by coliforms with growth at 35‡C and 45‡C increased after the storage period. The presence of
coliform organisms at 45‡C in samples prepared by group
B could only be detected after 6 h of storage. Moreover,
the isolation of Gram-negative bacilli occurred most frequently from the samples that had been previously maintained under refrigeration for 6 h.
Twenty-three IF samples, 17 prepared by group A and
six by group B, were contaminated with opportunistic
Gram-negative pathogens. Mixed £ora was frequently
found in IF and it was possible to identify up to ¢ve
di¡erent species from a single sample. Klebsiella pneumoniae, Citrobacter freundii, Cedacea davisae and Klebsiella
planticola were the predominant isolates from IF. P. aeruginosa was also present in IF samples analyzed (Table 2).
Susceptibility testing revealed that all the strains showed
4. Discussion
The role of food as a route for the circulation and
maintenance of opportunistic pathogens in hospitals and
as a possible source of nosocomial infections has been
described [2,5,8,14^16]. We found a statistically signi¢cant
di¡erence (P 6 0.05) in the rates of contaminated samples
prepared by the two groups of dietary technicians considering standards for colony counts and coliform growth at
35‡C and 45‡C, indicating that inadequate handling pro-
Table 2
Bacterial species isolated from IF samples
Species
Enterobacteriaceae
Klebsiella pneumoniae
Citrobacter freundii
Cedacea davisae
Klebsiella planticola
Enterobacter intermedium
Enterobacter cloacae
Cedacea neteri
Pantoea agglomerans
Kluyvera cryocrescens
Kluyvera ascorbata
Total
Pseudomonas aeruginosa
Number of isolates
48
27
22
21
15
11
5
4
2
1
156
17
Number of contaminated samples
% of contaminated samples
10
6
12
7
7
7
2
4
2
1
17
15
11.1
6.6
13.3
7.7
7.7
7.7
2.2
4.4
2.2
1.1
18.9
16.6
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L.A.M. Carneiro et al. / FEMS Microbiology Letters 228 (2003) 175^179
Table 3
Antimicrobial susceptibility pro¢les among Enterobacteriaceae and P. aeruginosa isolates (% resistant strains)
Species (number of strains)
AMC AMI
AMP CAZ
CFL CFO CIP
Enterobacter cloacae (n = 11)
Enterobacter intermedium (n = 15)
Pantoea agglomerans (n = 4)
Klebsiella pneumoniae (n = 48)
Klebsiella planticola (n = 21)
Citrobacter freundii (n = 27)
Cedacea davisae (n = 22)
Cedacea neteri (n = 5)
Kluyvera ascorbata (n = 2)
Kluyvera cryocrescens (n = 2)
Pseudomonas aeruginosa (n = 17)
72.7
93.3
100
14.5
9.5
48.1
95.4
100
100
100
ND
100
93.3
100
93.6
100
29.6
86.2
100
100
100
ND
81.8
93.3
100
29.1
9.5
55.5
95.4
100
100
100
ND
12.5
82.3
2.0
72.7
93.3
100
20.8
9.5
51.8
95.4
100
100
100
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
58.7
CLO
CTX
GEN
NET
PIP
SUT
9.1
9.1
16.6
18.6
70.5
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
41.1
9.1
16.6
100
4.1
88.3
94.1
TET
4.5
94.1
100
TIC
TOB
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
35.2
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
52.9
AMC, amoxicillin/clavulanic acid; AMI, amikacin; AMP, ampicillin ; CAZ, ceftazidime ; CFL, cephalotin; CFO, cefoxitin; CIP, cipro£oxacin ; CLO,
chloramphenicol; CTX, cefotaxime; GEN, gentamicin ; NET, netilmicin ; PIP, piperacillin ; SUT, sulfamethoxazole/trimethoprim; TET, tetracycline;
TIC, ticarcillin/clavulanic acid; TOB, tobramycin. ND, not determined.
All isolates were found to be susceptible to imipenem.
cedures may be an important source of contamination.
Since the two groups of technicians have the same training
background and should use the same procedures, we believe that the di¡erent levels of contamination between the
samples prepared by the two groups are due to poor personal hygienic conditions of some technicians and/or failure to observe the correct procedures.
An increase was observed in the rates of contamination
6 and 20 h after the preparation. The analysis of the di¡erent bacteriological indicators showed that not only the
number of samples unacceptable according to microbiological standards increased with time but also the concentration of microorganisms increased with the time between
the formula’s preparation and the time it was administered
to the patients. Similar variation was found with the MPN
of coliforms at 35‡C and 45‡C. Navajas et al. [4] reported
a four-fold variation between samples taken from IF immediately after the preparation and hours later. The time
lapse between the diet’s preparation and administration to
the patient constitutes an important factor as it favors the
multiplication of microorganisms. Even diets showing no
contamination in the kitchen could produce undesirable
e¡ects on reaching the patient’s bedside [4].
It was interesting to note that, in spite of the procedures
adopted, no E. coli strains were recovered from the milk
samples analyzed which suggests that there was no recent
fecal contamination of the formulas. On the other hand,
the most prevalent species were opportunistic pathogens
such as K. pneumoniae and C. freundii which are often
present in the hospital environment, suggesting contamination by nosocomial strains.
K. pneumoniae is one of the most important nosocomial
pathogens [17]. Colonization of newborns with multiresistant K. pneumoniae has been associated with arti¢cial feeding [6]. The environmental species K. planticola was the
fourth more frequently isolated microorganism from IF
samples in this study. Studies conducted in France and
Germany suggested that up to 19% of clinically identi¢ed
Klebsiella spp. belonged to the environmental species
K. planticola and K. terrigena. The authors also highlighted the occurrence of septicemia in neonates caused
by these species [18,19].
The substitution of normal £ora for K. pneumoniae has
been described following broad-spectrum antimicrobial
therapy [20]. K. pneumoniae has demonstrated the ability
to develop and/or acquire new resistance determinants and
is thought to be a reservoir for antimicrobial resistance
genes [17]. ESBL appear to be even more frequent in Latin
America where the prevalence of ESBL-producing strains
can reach up to 50% [21,22]. The ESBL-producing isolate
in this study was also resistant to cephalotin and ampicillin. In the NICU, this concern is augmented because of the
large amount of prophylactic antimicrobial drugs, including ESBL that these children receive. Further, 52.9% of
the P. aeruginosa demonstrated resistance to cipro£oxacin
and 94.1%, 82.3%, 70.5% and 52.9% showed resistance to
the aminoglycosides gentamicin, amikacin, netilmicin and
tobramycin, respectively. These drugs are widely used in
the treatment of P. aeruginosa infections [23], including
prophylactic and therapeutic usage in NICU [24].
The abuse of antibiotics creates a favorable condition
for the antimicrobial selection of resistant bacteria particularly related to long-term use of the L-lactams such as
amoxicillin/clavulanic acid, ampicillin, cephalotin and cefoxitin. Normally, when Gram-negative bacteria express
ESBL, they are susceptible to inactivation by L-lactamase
inhibitors (clavulanate). However, plasmid-encoded L-lactamases found in enteric organisms have also become resistant to inactivation by L-lactamase inhibitors [25].
The presence of bacterial species known to be important
nosocomial pathogens and their wide antimicrobial resistance pro¢les are reasons for concern considering the immunological immaturity of the population that will receive
these formulas. In addition, our results call attention to
the necessity of quality control programs and personnel
training in order to reduce the risk that this food might
FEMSLE 11247 12-11-03
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represent a vehicle of dissemination of opportunistic
pathogens carrying important resistance determinants.
[11]
Acknowledgements
[12]
The authors are grateful to Dr. J.A. Pereira and Dr.
E.A. Marques (Faculdade de Cie“ncias Me¤dicas), Dr.
Jose¤ L.B. Bandeira (chief of the Neonatal Intensive Care
Unit-HUPE) and S. Ferrara (chief of the lactary). This
work was ¢nancially supported by the FundacXa‹o de Amparo a' Pesquisa do Estado do Rio de Janeiro (FAPERJ),
Conselho Nacional de Desenvolvimento Cient|¤¢co e Tecnolo¤gico (CNPq) and CoordenacXa‹o de AperfeicXoamento
de Pessoal de N|¤vel Superior (CAPES).
[13]
[14]
[15]
[16]
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