TAXONOMY Back to top
The genera Bordetella, Achromobacter,
Alcaligenes, Kerstersia, and Advenella belong to the
family Alcaligenaceae(order Burkholderiales
in the β subclass of the Proteobacteria) (22).
Other members of this family
includeAzohydromonas, Brackiella, Castellaniella,
Derxia, Oligella, Pelistega, Pigmentiphaga,
Pusillimonas, Sutterella, andTaylorella (List of Prokaryotic Names
with Standing in
Nomenclature [http://www.bacterio.cict.fr/]). The genusBordetella contains eight
species: Bordetella avium, B. bronchiseptica,
B. hinzii, B. holmesii, B. parapertussis, B.
pertussis (the type species), B. petrii, and B. trematum (55,
66, 104, 105, 109, 116). Other
putative species, such as “B. ansorpii” and
other strains similar to B. trematum (45, 58),
have been described. B. pertussis, B.
parapertussis, and B. bronchiseptica could be
considered a single species, but chemotaxonomic
differences and differences in host range
and pathogenesis (66, 76)
support their status as separate species. Analysis of their genome
sequences revealed that B. parapertussis and
B. pertussis are independent derivatives of
a B. bronchiseptica-like ancestor (76).
The taxonomy of the genus Achromobacter is closely
intertwined with that of the genus Alcaligenes.
The genus Alcaligenes is now limited
to Alcaligenes aquatilis and Alcaligenes
faecalis (the type species) (20, 106),
while the
genus Achromobacter consists of six
species: Achromobacter denitrificans, A. insolitus, A.
piechaudii, A. ruhlandii, A. spanius, and A. xylosoxidans (the type species)
(20, 21, 105, 120). The genus Kerstersia was proposed for a
set of strains phenotypically
resembling A. faecalis that were classified
asKerstersia gyiorum or as belonging to at least
one other (so far unnamed) Kerstersia species
(20). Similarly, the genus Advenella was
created to harbor a number of Alcaligenes-like
strains; these strains belong either
toAdvenella incenata or to one of several
additional unnamed genomic species (22) or were
previously described as Tetrathiobacter species
(38).
DESCRIPTION OF THE GENERA Back to top
Bordetella
Bordetellae are small (1 to 2 μm), gram-negative,
nonsporulating coccoid rods (8). During
their adaptation to the human host, B.
pertussis and B. parapertussis underwent a reduction
in genome size (4.086 Mbp for B. pertussis, 4.774
Mbp for B. parapertussis, and 5.338 Mbp
for B. bronchiseptica). Insertion sequences
are found mainly in the genomes of B.
pertussis (IS481), B. parapertussis (IS1001), and B.
holmesii (IS481) and are found less so
in B. bronchiseptica (76,
92).
Bordetellae are catalase positive and oxidize
amino acids, but no carbohydrates can be
fermented. Some species possess peritrichous
flagella and are motile. Bordetellae are able to
grow in simple synthetic media under aerobic
conditions (except for B. petrii [see below]).
However, B. pertussis and B.
parapertussis are sensitive to toxic substances and metabolites
present in many microbiological media and need
special transport conditions, special culture
media, and prolonged incubation. The other species
are less sensitive and can be isolated by
routine microbiological procedures.
B. petrii is the most versatile Bordetella species, as it can grow
aerobically and anaerobically
and was initially found as a free-living
environmental bacterium. All other bordetellae are
found only in warm-blooded animals and humans. B.
avium is a pathogen for poultry and has
only once been isolated from humans (45).
B. bronchiseptica can cause respiratory infections
in many animal species and, infrequently, also in
humans. “B. ansorpii,” B. hinzii, B.
holmesii, B. petrii, and B. trematum are rarely found in
human infections and mainly cause
symptomatic diseases in immunocompromised
patients. B. parapertussis is found in sheep
and humans, and B.pertussis is thought to be a
strictly human pathogen (Table 1).
Bordetellae
express many virulence factors that are controlled by a complex virulence
expression
system operating in response to environmental conditions (BvgAS) (66).
In
subcultures,
these responses were called phases I, II, III, and IV, with phase I being
highly
pathogenic
and phase IV being almost apathogenic. The phases are now called mode X
(respiratory
tract infection), mode I (intermediate, possibly relevant for transmission),
and
mode
C (starvation). Virulence factors of bordetellae can be classified as adhesins,
autotransporters
(i.e., filamentous hemagglutinin [FHA], fimbriae [FIM], and pertactin
[PRN]),
and toxins (i.e., pertussis toxin [PT], adenylate cyclase toxin, and
lipopolysaccharide
[LPS])
(66). Only B. pertussis produces PT, encoded by the ptx gene,
whereas B.
parapertussis
(and B. bronchiseptica) contains the ptx gene
but normally lacks the promoter
(66).
PT has ADP-ribosyltransferase activity and ribosylates G proteins (66).
PT induces
lymphocytosis
and suppresses chemotaxis, oxidative responses, and the overall activity of
neutrophils
and macrophages. FHA is a large (220-kDa), surface-associated, secreted protein
and
mediates the adhesion of bordetellae to the ciliated epithelium of the upper
respiratory
tract.
FHA is produced by B. pertussis, B. parapertussis, and B.
bronchiseptica (66). FIM
types
2 and 3 (FIM2 and FIM3) represent the serotype-specific agglutinogens and are
important
factors in colonizing the respiratory mucosa. Isolates of B. pertussis can
display
FIM2,
FIM3, or both on their surface. Adenylate cyclase toxin is a hemolysin with
enzymatic
activity
(47). B. pertussis also produces an LPS without a repetitive
O-antigenic chain. PRN is
a
68- to 70-kDa surface protein that mediates eukaryotic cell binding in vitro (66).
PRN is
involved
in cell attachment by its Arg-Gly-Asp (RGD) motif and is highly immunogenic (48).
Achromobacter
Achromobacter
species are gram-negative, nonsporulating, straight rods of 0.8 to
1.2 by 2.5
to
3.0 μm. They are motile, with peritrichously arranged sheathed flagella; the
number of
flagella
varies from 1 to 20 per cell. They are strictly aerobic and nonfermentative,
although
strains
of some species are able to grow anaerobically with nitrate as an electron
acceptor
(120).
All Achromobacter species are oxidase and catalase positive, but none of
them
exhibits
urease, DNase, lysine decarboxylase, ornithine decarboxylase, arginine
dihydrolase,
or
gelatinase activity (21, 120) (Table 2). They grow well on simple media (including nutrient
agar),
and on nutrient agar, colonies are flat or slightly convex, with smooth
margins, and
range
from white to light brown (21). Under laboratory
conditions, growth occurs between
25
and 37°C and in the presence of 0 to 4.5% NaCl (21, 120). Achromobacter
species
contain
the Q-8 ubiquinone system (13). The predominant fatty
acids are C16:0 and C17:0
cyclo
(21). Although detailed information about the natural habitat of
these organisms is
lacking, soil and water are
considered to be primary sources of infection (13, 120).
Alcaligenes,
Kerstersia, and Advenella
Following
many taxonomic revisions, the genus Alcaligenes is now limited to A.
faecalis (the
type
species) andA. aquatilis. Within A. faecalis, three subspecies (A.
faecalis
subsp. faecalis, A. faecalis subsp. parafaecalis, and
A. faecalis subsp. phenolicus)
have
been described (84, 90). A. faecalis subsp. parafaecalis and A.
faecalis
subsp.phenolicus are represented by a single environmental
isolate each, and A.
aquatilis
strains have been recovered only from lake sediments (106).
Some A.
faecalis
strains produce a characteristic fruity odor and/or cause a
greenish discoloration of
blood
agar medium; these strains were previously referred to as “A.
odorans”
(54). Alcaligenes species are gram-negative, strictly aerobic
rods or coccobacilli
that
possess oxidase and catalase activity (13).
Cells are motile by means of 1 to 12
peritrichous
flagella (54). The optimum growth temperature is between 20 and 37°C. They
grow
well on simple media, and colonies on nutrient agar are generally nonpigmented.
The
predominant
fatty acids in Alcaligenes species are C16:0 and C17:0 cyclo (54,106).
Cells
of Kerstersia and Advenella species are gram- negative, small (1
to 2 μm), rod-shaped
or
coccoid cells and occur alone, in pairs, or in short chains. Motility is strain
dependent.
These
species grow well on simple media (including nutrient agar). On nutrient agar,
colonies
are
flat or slightly convex, with smooth margins, and range from white to light
brown. They
are
strictly aerobic and nonfermentative. All isolates studied so far are catalase
positive,
while
none of them exhibit β-galactosidase activity (20, 22). Kerstersia
strains can grow at
temperatures
between 28 and 42°C; growth also occurs with up to 4.5% NaCl. The
predominant
fatty acids inKerstersia species are C16:0 and C17:0 cyclo (20). Advenella
strains
can
grow at temperatures between 30 and 37°C and at NaCl concentrations between 0
and
3%.
The predominant fatty acids in Advenella species are C18:1 w7c, C16:0,
and C16:1 w7c (22).
EPIDEMIOLOGY AND TRANSMISSION Back
to top
B.
pertussis and B.
parapertussis
B.
pertussis and B. parapertussis cause pertussis, or whooping cough.
Infections by B.
parapertussis
tend to take a milder clinical course, with a shorter duration of
coughing and
less
vomiting and whooping (66, 112,117). B. pertussis continues to circulate in populations
where
high vaccination coverage of infants and children is achieved (79, 117),
because the
protection
induced after natural infection and vaccination wanes after several years
(79, 107).
In vaccinating countries, most cases of pertussis are now observed in neonates,
unvaccinated
young infants, older schoolchildren, adolescents, and adults (79, 111, 117). A
permanent
carrier state is not found in pertussis, although in outbreak situations
asymptomatic
transient carriage of BordetellaDNA detected by PCR has been observed in
up
to
~50% of individuals (66, 113). B. pertussis is transmitted by droplets, and in
susceptible
contacts
the transmission rate may be close to 90% (66).
In nonprimary cases, transmission
rates
are probably lower. Transmission of the disease in highly vaccinated
populations occurs
mainly
from adolescents and adults to infants or among older vaccinated children,
adolescents,
and adults (66, 117). Neonates and young infants are at greatest risk of being
infected
by their parents, although casual contacts may be important (115).
The continuing
circulation
of B. pertussis has prompted many industrialized countries to recommend
pertussis
vaccination with acellular pertussis vaccines for adolescents and adults, in
addition
to
children, in order to diminish the disease burden in these populations and to
reduce
morbidity
and mortality in newborns and young infants (79).
Other Bordetella Species, Achromobacter,
Alcaligenes,
Kerstersia, andAdvenella
Data
on epidemiology and transmission are limited to A. xylosoxidans infections
in cystic
fibrosis
(CF) patients. Persistent infections with this organism can occur, as
genotypically
identical
isolates are recovered from the respiratory tract over prolonged periods
(25, 51, 60).
There have been several reports of multiple CF patients being colonized or
infected
by the same A. xylosoxidans isolate (60, 80, 103).
However, no large-scale
outbreaks
caused by the same strain and involving multiple treatment centers have been
identified,
and epidemiological studies revealed that there are many different A.
xylosoxidans
strains infecting CF patients.
CLINICAL SIGNIFICANCE Back
to top
B.
pertussis and B.
parapertussis
After
an incubation period of 7 to 10 days (range, 4 to 28 days), the primary
infection starts,
with
rhinorrhea, sneezing, and nonspecific coughs (catarrhal phase). The typical
clinical
symptoms
of pertussis are found in primary infections of nonvaccinated children and
include
coughing
spasms, whooping, and vomiting (paroxysmal phase) (77).
Cases in neonates and
unvaccinated
young infants often present with apnea as the only symptom (66, 79).
In older
schoolchildren,
adolescents, and adults, the symptoms can vary widely. Adult pertussis is
associated
with a long illness, and the persistent cough is often paroxysmal and has a
mean
duration
of approximately 6 weeks. It is frequently accompanied by choking, vomiting,
and
whooping
(117). The CDC clinical case definition for pertussis
(http://www.cdc.gov/vaccines/pubs/surv-manual/chpt10-pertussis.html#case)
requires 14
days
of coughing with paroxysms, whooping, or vomiting. The disease is most
dangerous in
infants,
and most hospitalizations and deaths occur in this age group. Fatal cases of
the
disease
may go undetected in young infants (75, 79).
Pertussis-like
symptoms may also be caused by adenoviruses, respiratory syncytial virus,
human
parainfluenza viruses, influenza viruses, Mycoplasma pneumoniae, and
other agents
(79).
Coinfections of B. pertussis and respiratory syncytial virus are
observed frequently in
infants
(23).
Other Bordetella Species
B.
bronchiseptica (69, 81), B. holmesii (116),
and B. hinzii (4, 34, 49, 52) can rarely be
isolated
from respiratory materials from patients with pertussis-like symptoms and other
respiratory
symptoms. In many cases, patients are systemically or locally
immunocompromised,
such as human immunodeficiency virus-infected patients or patients
suffering
from CF (19, 96, 97). As with other gram-negative nonfermentative bacilli, rare
cases
of bacteremia and septicemia have been described.
B.
trematum (24, 104) has been isolated from people working with poultry, and “B.
ansorpii”
(33, 58) is another rare cause of septicemia. B. petrii (32, 98),
and possibly other
environmental
bordetellae (110), is rarely found in clinical material, and B. avium has
so far
been
isolated from respiratory material from humans only once (45) (Table
1).
Achromobacter
All Achromobacter
species except A. ruhlandii have been recovered from clinical
samples or
from
the hospital environment. A. xylosoxidans (previously known as A.
xylosoxidans
subsp. xylosoxidans) is an opportunistic human pathogen
capable of causing a
wide
range of infections, such as bacteremia, meningitis, pneumonia, and peritonitis
(1, 40).
It
has also been involved in nosocomial infections attributed to contaminated
disinfectants,
dialysis
fluids, saline solution, and water (70). A.
xylosoxidans has been reported from CF
patients
since 1985, and prevalence rates in CF patients vary from 3 to 18%
(12, 25, 51, 96). A.
xylosoxidansinfections in CF patients do not seem to have a significant
impact
on lung function (25, 80, 87). A. piechaudiihas been isolated from various clinical
samples,
including pharyngeal swabs, the nose, wounds, blood, and chronic ear discharge
(56).
There is a single report of recurrent A. piechaudii bacteremia
associated with an
intravenous
catheter in an immunocompromised patient (53). A.
denitrificans (previously
known
as A. xylosoxidans subsp. denitrificans) has been recovered from
many clinical
specimens,
such as urine, proctoscopy specimens, prostate secretions, the buccal cavity,
pleural
fluid, and eye swabs (54), but there are no detailed reports about its clinical
significance.
A. insolitus (in a leg wound and in urine) and A. spanius (in
blood) have been
found
in clinical material, but their significance is unclear (21).
Alcaligenes,
Kerstersia, and Advenella
A.
faecalis strains have been isolated from a wide range of clinical samples (54),
but the
accuracy
of the identification (especially in older reports) is difficult to assess. A.
faecalis was
found
in cases of bacteremia following surgery or cancer treatment, ocular
infections, a
pancreatic
abscess, infections following bone fractures, urine, and ear discharge (1, 6).
There
are
sporadic reports of the recovery of A. faecalis in sputa of CF patients
(114). K.
gyiorum
was isolated from human feces, leg wounds, and sputum (20). A.
incenata has been
recovered
from human sputum (including sputa from CF patients) and blood, and several
unnamedAdvenella
species were isolated from similar sources (22).
COLLECTION, TRANSPORT, AND STORAGE OF
SPECIMENS Back to top
B.
pertussis and B.
parapertussis
Sampling
for culture and PCR is difficult and markedly influences the sensitivity of
these
tests.
Nasopharyngeal aspirates are adequate samples for infants and young children,
and
for
culture, they are more sensitive than swabs (43).
Nasopharyngeal swabs taken by
trained
personnel from older children, adolescents, and adults provide valid specimens
from
these
age groups. Nasopharyngeal swabs should be taken by gently inserting the swab
into
the
nasopharynx under the inferior nasal choana, and the nose of the patient should
be bent
slightly
upwards. If possible, two nasopharyngeal swabs should be taken, with one taken
from
each nostril. Swabs should be small and made of Dacron or rayon.
Calcium-alginate
swabs
and swabs with aluminum shafts should not be used for PCR (86).
Flocked nylon
swabs,
which are more convenient for the patient, may also be used but have not been
validated
for B. pertussis PCR or culture. Samples should be taken before
antibiotic
treatment
is started.
The
most sensitive method for culture is direct plating and preincubation at 35 to
37°C for 24
h
before transport (73). Transport time is critical, and a transport medium protecting
the
bacteria
from drying is required. Bacteriological transport media such as Casamino Acids
or
Amies
medium with charcoal may be used, but transport time should not exceed 48 h.
Halfstrength
Regan-Lowe
(RL) charcoal-blood medium is also used for transport. In contrast to
other
transport media, RL medium can serve as an enrichment medium for B.
pertussis.
Transport
at 4°C increases culture positivity but adds to logistical problems (73).
For PCR,
swabs
can be transported dry at ambient temperature. The use of microbiological
transport
media
such as Amies medium with charcoal does not interfere with PCR (86).
Other
respiratory
samples, such as throat swabs, sputum samples, or throat washes, are less
suitable
and have not been validated (66, 73).
Other Bordetella Species, Achromobacter,
Alcaligenes,
Kerstersia, andAdvenella
For
other bordetellae, normal microbiological transport media seem to be suitable
for
transport.
Similarly,Achromobacter, Alcaligenes, Kerstersia, and Advenella species
can
survive
in a wide range of environments and at various temperatures. Standard
collection,
transport,
and storage techniques are sufficient to ensure recovery of these organisms
from
clinical
specimens, contaminated nosocomial sources, and the environment.
DIRECT DETECTION METHODS Back
to top
B.
pertussis and B.
parapertussis
DFA
Direct
fluorescent-antibody staining (DFA) requires nasopharyngeal swabs or
nasopharyngeal
aspirates;
it is rapid and simple but lacks sensitivity and specificity (66).
Antibodies for DFA
are
mostly polyclonal or directed against the LPS of B. pertussis. DFA is
not accepted as
proof
of infection in notifying countries (73).
PCR
Depending
on age, vaccination status, and duration of symptoms of the patients, PCR is
between
twofold and sixfold more sensitive than culture (35, 86).
Block-based and real-time
PCR
methods seem to have comparable sensitivities (74, 86, 100).
Similar to the case for
culture,
the sensitivity of PCR decreases with the duration of coughing; however, due to
its
higher
sensitivity, it may be a useful tool for diagnosis for up to 4 to 6 weeks of
coughing
(86).
Real-time PCR formats have the advantage of offering a result within several
hours.
DNA
extraction is necessary to limit inhibition of PCR (86).
Commercially available extraction
kits
seem to be comparable and appropriate (86),
but no head-to-head comparison has yet
been
done. These kits are not FDA cleared or CE marked for this purpose. Most
laboratories
use
the IS481 target (copy number, ~200 per cell) (63, 86)
for the detection of B.
pertussis
and the IS1001 target (copy number, ~20 per cell) (41)
for the detection of B.
parapertussis.
There has been concern about the specificity of detection of B. pertussis due
to
sequence identity of IS481 with B. holmesii (63, 85).
Clinical samples were retested using
primer-probe
sets specific for B. holmesii DNA (3),
and B. holmesii-specific sequences were
not
detected in any of the retested samples. These results suggest that IS481 assays
may
currently
be sufficiently specific for the laboratory diagnosis of B. pertussis. However,
the
periodic
appearance of B. holmesii in some host populations and a possible
carriage of
IS481
by some strains of B. parapertussis and B. bronchiseptica make
it necessary to
monitor
the specificity of IS481-based assays. No specificity problems were
reported for the
detection
of IS1001 to diagnose B. parapertussis infections, although B.
holmesii shares
some
sequence identity (86). The PT promoter (ptxA-Pr) is another target for B.
pertussisspecific
PCR
assays (31, 35, 99), whereas the detection of the PT gene will detect both B.
pertussis
and B. parapertussis. Amplification of targets in the FHA
gene, the PRN gene, and
the
porin gene was also used for detection of B. pertussis (83).
Tests detecting one-copy
genes
were, however, consistently less sensitive than IS481-based PCRs.
Detection can be
done
sequence specifically by use of fluorescence resonance energy transfer
hybridization
probes,
TaqMan probes, and molecular beacons and also by non-sequence-specific formats
using
Sybr green I (86). For specificity reasons, most laboratories use
sequence-specific
formats
(86). Duplex PCRs for B. pertussis (B. holmesii) andB.
parapertussis have been
developed.
Commercial multiplex PCRs for the detection of various respiratory agents,
including
bordetellae, are available. External quality control programs have been
implemented
in European countries (74). In outbreak situations and after household
contacts,
a positive PCR result may also be found for patients with very few or no
symptoms
(16, 113).
ISOLATION PROCEDURES Back
to top
B.
pertussis and B.
parapertussis
Culture
is thought to be almost 100% specific, because very rarely have patients been
found
to
harbor B. pertussis without any symptoms (73).
Several culture media, such as RL
medium
(73), Bordet-Gengou (BG) medium (8, 73),
and Stainer-Scholte medium, have been
used
for culture of B. pertussis and B. parapertussis. RL medium is
made with casein digest,
beef
extract, starch, and charcoal medium supplemented with horse blood. BG medium
consists
of potato infusion with glycerol and horse blood or sheep blood.
Stainer-Scholte
medium
is a fully synthetic blood-free medium often used in vaccine production (73).
Most
media
are supplemented with cephalexin to suppress concomitant bacteria. RL medium
can
be
stored for 4 to 8 weeks, and BG medium has a shelf life of 5 days. Incubation
time should
be
at least 1 week at 35 to 37°C at ambient atmosphere. The sensitivity of culture
depends
on
the duration of symptoms and the age and vaccination status of the patient, and
it varies
between~60%
for young unvaccinated infants with symptoms of a few days in duration and
<5% for adolescents and
adults with more than 3 weeks of coughing (117).
Achromobacter
species can be isolated from clinical samples by the use of simple
media and
a
selective enteric medium, such as MacConkey agar (54).
It has been reported that a
minority
of A. xylosoxidans isolates grow on Burkholderia cepacia-selective
oxidativefermentative-
polymyxin
B-bacitracin-lactose agar or Pseudomonas cepacia agar (30% and
20%,
respectively) but do not grow on Burkholderia cepacia-selective agar (46).
Recent data
from
the U.S. CFF Burkholderia cepacia Research Laboratory and Repository (J.
J. LiPuma,
unpublished
data) and a previous study (118) indicate that the majority
of A.
xylosoxidans
isolates (~60%) will grow onBurkholderia cepacia-selective
agar. Results
obtained
in a small-scale study suggest that particular selective media, such as
gramnegative
organism-selective
agar, may increase the recovery of A. xylosoxidans from CF
sputa
(71).
IDENTIFICATION Back to top
B.
pertussis and B.
parapertussis
B.
pertussis colonies become visible after 3 to 7 days of incubation, and B.
parapertussis
colonies are visible already after 2 to 3 days. On RL medium,
colonies are very
small,
round, and domed and appear silvery (Fig. 1). B.
parapertussis colonies are larger and
less
shiny. The minute colonies on BG medium have a small zone of beta-hemolysis. B.
pertussis
is a small, coccoid, gram-negative rod which is catalase and
oxidase positive (B.
parapertussis
is oxidase negative). The identities of these two species can best
be confirmed
by
agglutination with specific antibodies. Further biochemical characteristics are
given
in Table
3. Due to the fastidious growth of B. pertussis and B.
parapertussis, commercial
systems
for gram-negative rods are not reliable for identifying these species. 16S rRNA
gene
sequencing
(69) and matrix-assisted laser desorption ionization–time-of-flight
mass
spectrometry (MALDI-TOF) (26)
can be applied effectively for identification.
Other Bordetellae
Colony morphology is not discriminative, and the
bacteria are small coccoid rods that are
catalase positive. In most instances, a biochemical
system for identification of gram-negative
bacilli will be used, such as API-NE, Vitek
(bioMerieux), MicroScan (Siemens), Phoenix
(Becton Dickinson) (94), and other systems.
These systems were validated for
nonfermentative rods, among which a few Bordetella
spp. were also evaluated. Overall, the
specificity of these biochemical systems for these
bacteria is not very high, and thus an
algorithm was recently proposed for the API-NE and
Vitek II systems that uses 16S rRNA
gene sequencing if the results of the biochemical
identification are not reported as
“excellent” or “very good” (17).
16S rRNA gene sequencing offers more reliable identification
results and is available in many reference
laboratories (10, 30, 82, 122). MALDI-TOF offers
an alternative to sequencing (26).
Apart from B. bronchiseptica, isolation of other bordetellae
from clinical material is a rare event, and their
identification might be confirmed by a
reference laboratory.
Achromobacter
Achromobacter species typically show very limited action on carbohydrates (54),
which
hampers accurate identification at the genus level
based on biochemical characteristics.
Thus, 16S rRNA gene sequence analysis is
recommended (13). Biochemical characteristics
that distinguish the various Achromobacter species
and discern them from A. faecalis, K.
gyiorum, and A. incenata are shown in Table 2. Several commercial
systems allow the
identification of Achromobacter species. A
comparison indicated that A. xylosoxidans was
correctly identified in 88%, 71%, 54%, and 21% of
cases, using the RapID NF Plus, API
Rapid NFT, Vitek, and Remel systems, respectively
(57). A. xylosoxidans may be
misidentified as a member of the B. cepacia complex
(and the other way around) by some
commercial systems (93, 122),
and due to its weak biochemical reactivity, prolonged
incubation (e.g., for up to 72 h with the API 20
NE system) may be required to obtain a
reliable identification. A PCR assay (based on the
16S rRNA gene) was developed for A.
xylosoxidans, but positive results may also be obtained with strains of
other Achromobacter species as well as with
some Bordetellaspecies (62, 97). The use of
fluorescent in situ hybridization (FISH) with a
probe directed against the 16S rRNA gene has
been reported for the identification of A.
xylosoxidans (114). FISH assays had a high
sensitivity and better specificity than the PCR
assay (62, 97, 114), although cross-reactivity
with A. ruhlandii and aChryseobacterium sp.
isolate was observed (114). MALDI-TOF and
Fourier transform infrared spectroscopy have also
been used successfully to identify A.
xylosoxidans (9, 26).
Alcaligenes, Kerstersia, and Advenella
For Alcaligenes, Advenella, and Kerstersia,
16S rRNA gene sequence analysis is
recommended for accurate identification at the genus
level (13). Differential biochemical
reactions are listed in Table 2. Members of the genus Advenella can be separated from
related species by their inability to assimilate
phenyl acetate.Kerstersia strains are oxidase
negative. A distinguishing characteristic of A.
faecalis isolates is that they reduce nitrite but
not nitrate. Molecular techniques have not yet
been developed for these organisms, with the
exception of a 16S rRNA gene-directed FISH probe
for A. faecalis (114).
TYPING SYSTEMS Back to top
B. pertussis and B. parapertussis show only a very small amount of
genomic heterogeneity
(11). The ptx gene and the prn genes
are polymorphic in the B. pertussis genome, and
various ptx and prn types (prn1 to
prn8) have been identified (102). The expression of
fimbriae undergoes temporal changes, possibly
influenced by vaccine coverage (102).
Circulating isolates of B. pertussis have
been typed by various methods, such as pulsed-field
gel electrophoresis (PFGE), analysis of
variable-number tandem repeats, restriction fragment
length polymorphism analysis (102),
and others (82). The prn types of clinical isolates
mostly differ from theprn type of the
currently used vaccine, but so far no changes in the
effectiveness of acellular vaccines have been
observed. Studies using PFGE have shown that
the overall genomic heterogeneity of clinical
isolates decreases (44).
Typing of A. xylosoxidans isolates by PFGE
of fragments obtained after digestion with XbaI,
SpeI, or DraI has been used in several studies (69,
80) and is reported to have a high
discriminatory power. Randomly amplified
polymorphic DNA PCR (60) and PCR with
enterobacterial repetitive intergenic consensus or
repetitive extragenic palindromic primers
(103) have also been used. The discriminatory power of
ribotyping (using the Riboprinter
microbial characterization system) was rather low
(18). Selective restriction fragment
amplification (using EcoRI and MseI) was also
successfully applied to A. xylosoxidans (103).
SEROLOGIC TESTS Back to top
B. pertussis and B. parapertussis
Pertussis in older vaccinated children, in
adolescents, and in adults is mostly diagnosed by
serological tests. The use of enzyme-linked
immunosorbent assay (24, 28, 37, 64) to
quantify anti-PT antibody levels is a validated
and sensitive diagnostic technique and can be
performed with paired (acute- and
convalescent-phase samples) or single serum samples
(73). Paired-sample serology is a standardized method
of diagnosis (61), but single-sample
serology also provides good sensitivity and
specificity to determine cases in older children,
adolescents, and adults (5,
15, 27, 121). A WHO reference preparation for human pertussis
serology is available (119),
and quantitative results of pertussis serology should be reported
in international units/milliliter. Immunoglobulin
G (IgG) anti-PT antibodies at >100 to 125
IU/ml can be used as an indicator of recent
contact with PT-producing bacteria (67, 78,
108).
IgG antibodies are those mostly measured, but the
roles of other isotypes, such as IgA and
IgM, are not clear (66). Serology cannot
distinguish between vaccine- and infection-induced
immunological responses (symptomatic or
asymptomatic infection) (66, 101). Commercial
assays are of very variable quality (85a)
and are in need of further standardization (101).
Pertussis serology may not be used for 1 year
after vaccination with acellular vaccines.
ANTIMICROBIAL SUSCEPTIBILITY Back to top
B. pertussis and B. parapertussis
B. pertussis and B. parapertussis are susceptible in vitro to a range of
antibiotics, including
penicillins, macrolides, ketolides, quinolones,
and other antibiotics, including tetracyclines,
chloramphenicol, and trimethoprim-sulfameth
oxazole, whereas they are resistant to most
oral cephalosporins (66,
73). However, in contrast to the case for other
bacterial diseases,
the exact relationship between the
pharmacokinetics and pharmacodynamics of these
antibiotics and the in vitro susceptibility of the
organism is unknown. Furthermore, the effect
on the symptoms of pertussis is not well
documented (2, 79).
Methods for antibiotic sensitivity testing of B.
pertussis and B. parapertussis are not
standardized. If testing is done, the methods
include broth macro- and microdilution
methods, agar dilution methods, breakpoint methods,
and Etest, whereas the disk diffusion
method is mostly not feasible (116).
Erythromycin resistance may be evaluated by the disk
diffusion method. Erythromycin resistance was
documented first in the United States and
subsequently in other countries. In retrospect,
erythromycin-resistant organisms were found
in strain collections from the 1960s, and no data
so far suggest that this resistance is
spreading (116). Erythromycin resistance
is mediated by a mutation in the macrolide binding
domain of the 23S rRNA. Routine antibiotic
susceptibility testing of B. pertussis isolates is not
recommended and should only be done when special
clinical or epidemiological
circumstances are found (81).
Continued surveillance of these isolates, when performed,
should also include antimicrobial susceptibility
testing.
Other Bordetellae
B. bronchiseptica possesses a β-lactamase (50)
and is resistant to many penicillins and
cephalosporins and mostly resistant to
trimethoprim-sulfamethoxazole (72). A recent study
of canine and feline isolates (91)
showed that most isolates were sensitive to amoxicillinclavulanic
acid, tetracycline, gentamicin, and a quinolone. B.
avium was resistant to
cefuroxime, trimethoprim-sulfamethoxazole, and
tetracycline and sensitive to ampicillin,
mezlocillin, and gentamicin (72).
A human B. hinzii isolate was sensitive to amoxicillin,
gentamicin, and meropenem but resistant to
cefuroxime, ceftriaxone, and ciprofloxacin (34).
A human “B. ansorpii” isolate was resistant
to aztreonam, cefuroxime, and ceftriaxone and
sensitive to amoxicillin, gentamicin, and
ciprofloxacin (33). Antimicrobial sensitivity testing of
these Bordetella isolates should be
interpreted in accordance with criteria for other
infrequently isolated and fastidious nonfermentative
gram-negative rods.
Achromobacter
Methods for antibiotic sensitivity testing of Achromobacter,
Alcaligenes,
Kerstersia, and Advenella species are not standardized. If testing is
done, the methods
include broth macro- and microdilution, agar dilution
methods, breakpoint methods, and
Etest.
A. xylosoxidans isolates (39) were sensitive only to imipenem, piperacillin,
ticarcillinclavulanic
acid, ceftazidime, and
trimethoprim-sulfamethoxazole. Aminoglycosides,
expanded-spectrum cephalosporins other than
ceftazidime, and quinolones showed no
activity. The majority of the strains were
resistant to a conventional tobramycin
concentration, but 41% of the strains were
inhibited by the higher tobramycin concentrations
achievable by aerosol delivery of the antibiotic.
Similarly, 92% of strains were inhibited by
high doses of colistin (100 μg/ml). Little
synergistic activity was measured for combinations
of antibiotics, and additive activity was noted
with chloramphenicol-minocycline,
ciprofloxacin-imipenem, and
ciprofloxacin-meropenem (89). A. xylosoxidans was resistant to
azithromycin and clarithromycin, and only modest
synergistic and/or additive activities were
observed when azithromycin was combined with
meropenem or trimethoprimsulfamethoxazole
(88). These data correlate well with many smaller
clinical studies and case
reports (51, 65,80,
103). Antimicrobial susceptibility data for other Achromobacter
species
are rare, but A. spanius and A.
insolitus were resistant to most quinolones, macrolides, and
cephalosporins tested (22),
while a blood isolate of A. piechaudii was resistant to ampicillin,
cefpodoxime, and gentamicin but susceptible to all
other antibiotics tested (53).
Alcaligenes, Kerstersia, and Advenella
A. faecalis is more susceptible to antibiotics than A. xylosoxidans (95).
Most A.
faecalis strains are resistant to amoxicillin, ticarcillin, aztreonam,
kanamycin, gentamicin,
and nalidixic acid, while being susceptible to the
combination of amoxicillin or ticarcillin with
clavulanic acid, to various cephalosporins, and to
ciprofloxacin (7). Most Kerstersia isolates
are susceptible to ciprofloxacin and cefotaxime (20),
and antimicrobial susceptibility
inAdvenella spp. has not yet been studied.
EVALUATION, INTERPRETATION, AND REPORTING OF
RESULTS Back to top
Due to its sensitivity and speed, PCR is the
preferred method for the direct detection of B.
pertussis and B. parapertussis. A positive IS481 PCR from a
nasopharyngeal swab or a
nasopharyngeal aspirate can be considered to
indicate a B. pertussis (or B. holmesii)
infection when the clinical symptoms are in
accordance with this result. The specificity may
be substantiated by a positive ptxA-Pr PCR
by reference laboratories. Due to the higher
sensitivity of IS481 PCRs, a few samples
may be positive for IS481 and negative for ptx-Pr
(35). These results can be reported as Bordetella DNA
positive (B. pertussis, B.
holmesii, or B. bronchiseptica). A positive IS1001 PCR result
from a nasopharyngeal swab or
a nasopharyngeal aspirate is indicative of B.
parapertussis infection, without further tests.
If culture is performed, the isolation of B.
pertussis and B. parapertussis implies an infection,
although the sensitivity is sufficiently high only
for neonates and unvaccinated infants.
Routine antimicrobial sensitivity testing is not
necessary.
Serological diagnosis of pertussis is usually
based on single-sample serology. Results cannot
be interpreted correctly for about 1 year after
vaccination with acellular pertussis vaccines.
An IgG anti-PT titer of ≥100 to 125 IU/ml is
mostly used as an indicator of recent contact. In
adolescent and adult populations, an IgG anti-PT
titer of <40 IU/ml may be interpreted as
not indicative of recent infection. Apart from
Massachusetts, serology is not accepted as a
confirmation of cases in other U.S. states. Many
European countries with statutory
notification and laboratory confirmation accept
serology as proof of infection.
Similar to the case for other rarely isolated
gram-negative nonenteric rods, the clinical
relevance of otherBordetella spp., Achromobacter
spp., Alcaligenes spp., Kerstersia spp.,
and Advenella spp. isolated from clinical
material should be discussed on a case-to-case
basis between the microbiology laboratory and the
clinician. Antimicrobial testing of these
species should be performed and can be helpful in guiding
therapeutic decisions.
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