多重藥物或現有全部藥物抗藥性Acinetobacter baumannii引起之院內感染：臨床表現、預後、菌株之藥物感受性

行政院國家科學委員會專題研究計畫 成果報告

多重藥物或現有全部藥物抗藥性 Acinetobacter baumannii

引起之院內感染：臨床表現、預後、菌株之藥物感受性

中

華

民

國 92 年 10 月 6 日

Dissemination of A Clone of Unusual Phenotype of

Pandr ug-Resistant Acinetobacter baumannii at a Univer sity

Hospital in Taiwan

LU-CHENG KUO, 1 LEE-JENE TENG,1, 3 CHONG-JEN YU, 1

SHEN-WU HO,1, 3ANDPO-REN HSUEH1, 2 *

Departments of Internal Medicine1and, Laboratory Medicine 2

National Taiwan University Hospital; School of Medical Technology, 3National Taiwan University College of Medicine, Taipei, Taiwan

Running head: PANDRUG-RESISTANT A. BAUMANNII WITH UNUSUAL

PHENOTYPE

*Corresponding author. Mailing address: Departments of Laboratory Medicine and

Internal Medicine, National Taiwan University Hospital, No 7, Chung-Shan South

Road, Taipei, Taiwan. Phone: 886-2-23123456 ext. 5363. Fax: 886-2-23224263.

ABSTRACT

Fr om December 2002 to Febr uar y 2003, 15 isolates of pandr ug-r esistant

unidentified Acinetobacter species wer e r ecover ed fr om seven patients with

nosocomial infections or colonizations tr eated at differ ent war ds or intensive

car e units at the National Taiwan Univer sity Hospital. These isolates, which wer e

glucose- and lactose-non-acidifier s, failed to r ecognize to the species level using

thr ee commer cial identification systems: the Vitek GNI, API 20NE system

(bioMer ieux, Mar cy L’Etoile, Fr ance) and the Phoenix System

(Becton-Dickinson, Spar ks, Md.), and 16S r RNA gene sequence analysis.

However , 16S-23S r RNA inter genic spacer PCR-r estr iction fr agment length

polymor phism pr ofiles and the sequence analysis of these isolates both identified

as A. baumannii. All these isolates wer e unifor mly r esistant to

ampicillin-sulbactam (MICs, 128->128 µg/ml), ceftazidime (MICs, 64->128 µg/ml), piperacillin-tazobactam (MICs, 128->128 µg/ml), cefepime (MICs, 16-32 µg/ml), aztreonam (MICs, 64-128 µg/ml), ciprofloxacin (MICs, 64-128 µg/ml), tr ovafloxacin (MICs, 8-16 µg/ml), moxifloxacin (MICs, 4 µg/ml), garenoxacin (MICs, 16-32 µg/ml), amikacin (MICs, >128 µg/ml), imipenem (MICs, 8-16 µg/ml), and meropenem (MICs, 128->128 µg/ml). The identity of the pulsed-field

gel electr ophor esis patter ns and antibiotypes among these isolates fr om the same

patients with an inter val of 4-8 weeks and differ ent patients indicated that this

pandr ug-r esistant A.baumannii with unusual phenotype could have long-ter m

INTRODUCTION

Bacteria that constitute the genus Acinetobacter were originally identified in the

early twentieth century, but it was appreciated as a ubiquitous pathogen only in the

last decade (2). Acinetobacter species are aerobic, encapsulated, nonmotile, and

gram-negative organisms. Among them, A. baumannii is the species most commonly

involved in infections. Nosocomial infections caused by multidrug-resistant A.

baumannii have been reported in recent years (4, 5, 12, 13, 24). The emergence of

carbapenem-resistant A. baumannii (CRAB) was reported in the United States in 1991

(8). Since then, CRAB infections and hospital-wide outbreaks have been reported

worldwide (1, 5). Isolates of pandrug-resistant A. baumannii (PDRAB), which was

resistant to all commercially available antibiotics, were first recovered in May 1998 at

the National Taiwan University Hospital (NTUH) (14). Since then, clusters of

PDRAB infections and nosocomial outbreaks persisted, although the incidence of

nosocomial infections caused by PDRAB declined in the past two years (12, 13, 15).

In December 2002, an isolate of PDR Acinetobacter species was recovered from

respiratory secretions of a hospitalized patient. This isolate was identified

presumptively to be an unusual phenotype of PDR A. baumannii (PDRABup) because

of its negative reaction to 10% lactose. Further biochemical profile studies using three

then, a total of 15 isolates of this unusual phenotype of PDR Acinetobacter strains

were recovered various clinical specimens of seven hospitalized patients in the

following three months at the hospital. The aim of this study is to determine the

species level of this unusual phenotype of Acinetobacter species, their in vitro

PATIENTS AND METHODS

Clinical data. From 1 December 2002 through 28 February 2003, 15 isolates of

PDRABup recovered from various clinical specimens of seven patients who were

treated at the National Taiwan University Hospital were identified. These isolates

were resistant to all commercially available antibiotics tested (i.e.

ampicillin-sulbactam, ceftazidime, piperacillin-tazobactam, cefepime, aztreonam,

ciprofloxacin, trovafloxacin, moxifloxacin, garenoxacin, amikacin, imipenem, and

meropenem). Relevant information on the clinical presentation of these patients was

collected. These data included the underlying diseases, associated medical condition

(the use of an indwelling catheter and administration of chemotherapy), clinical

syndromes, days of positive culture for this organism after hospitalization, preceding

antibiotic regimens before acquisition of this organism, and outcome.

Identification of bacter ial isolates. The 15 isolates were initially identified as

PDRABup on the basis of colonial morphotypes, gram staining characteristics,

oxidase reaction, and growth on triple-sugar agar. These isolates were intended to

identify to species level by their biochemical profiles obtained with the Vitek GNI and

API 20NE system (bioMerieux, Marcy L’Etoile, France) and Phoenix System

(Becton-Dickinson, Sparks, Md.).

amplification product was performed according to previous description. PCR

amplification of 16S-23S intergenic spacer sequences (ITS) was also performed and

the amplicons were digested by AluI and then subjected to restriction fragment length

polymorphism (RFLP) analysis and direct sequencing study based on the protocol

described previously.

Antimicrobial susceptibility testing. The minimum inhibitory concentrations

(MICs) of antimicrobial agents for the 15 isolates of PDRABup were determined

using the agar dilution method according to guidelines established by the National

Committee for Clinical Laboratory Standards (NCCLS) (21, 22). The following 14

antimicrobial agents were obtained as standard reference powders of known potency

for laboratory use: ampicillin-sulbactam, and trovafloxacin (Pfizer Inc., New York,

NY); cefotaxime (Aventis Pharma, Romainville, France); ceftazidime (Glaxo,

Greenford, U.K.); flomoxef (Shiohogi & Co., Ltd. Osaka, Japan); aztreonam,

cefepime, amikacin, and garenoxacin (Bristol-Myers Squibb, Princeton, N.J.);

piperacillin-tazobactam (Wyeth-Ayerst Laboratories, Pearl River, N.Y.); imipenem

(Merck Sharp & Dome, Rahway, N.J.); meropenem (Sumitomo Pharmaceuticals,

Osaka, Japan); and ciprofloxacin and moxifloxacin (Bayer Co., West Haven, Conn.).

The isolates were grown overnight on trypticase soy agar plates supplemented with

inocula were prepared by suspending the freshly grown bacteria in sterile normal

saline and adjusting to a 0.5 McFarland standard. Using a Steers replicator, an

organism density of 104 CFU/spot was inoculated onto the unsupplemented

Mueller-Hinton agar (BBL Microbiology Systems) with various concentrations of

antimicrobial agents and then incubated at 35oC in ambient air.

For synergy analysis, five pairs of antimicrobial disks (ceftazidime and amikacin,

cefepime and amikacin, imipenem and amikacin, imipenem and

ampicillin-clavulanate, and imipenem-ciprofloxacin) were applied onto

unsupplemented Mueller-Hinton agar with a distance of 15 mm (center-to-center) of

two disks. Synergy between two antimicrobial agents was identified if the presence of

enhanced inhibition zone between two antimicrobial disks.

Molecular typing. Genotypes of the 15 isolates of PDRABup and five PDRAB

isolates, one each from clones 1 to 5 reported previously (14), were determined by the

PFGE. For PFGE, DNA extraction and purification were also carried out as described

previously (14). The DNA was digested by the restriction enzyme SfiI and the

restriction fragments were separated in a CHEF-DRIII unit (Bio-Rad, Hercules,

Calif.). Interpretation of the PFGE profiles followed the description by Tenover, et al.

(26) PFGE profiles of the isolates were considered derived from a common ancestor

Definitions. Antibiotypes were considered identical if the MICs of all

antimicrobial agents tested were identical or within a 1-dilution discrepancy. Isolates

were defined as the same strain or originating from a single clone if they had identical

RESULTS

Char acter istics of the patients. The clinical characteristics of the 7 patients

with infections caused by PDRABup are provided in Table 1. Their mean age was 64

years old (range, 14-90 years). Three patients (43%) were male. One patient had

underlying malignancy (cholangiocarcinoma) and none had hematological

malignancies or immunodeficiency. All except one patient (patient 5) had fever as the

presentation of the infection. Six patients were in bed-ridden state due to stroke or

head injury. All except patients 2 and 5 received endotracheal mechanical ventilation.

All infections or colonizations due to PDRABup were all hospital-acquired, occurring

9 to 38 days after admission.

The most common source of infection was respiratory tract. PDRABup was

isolated from multiple sites in three patients (patients 3, 4, and 7). Co-isolates were

identified from the same specimen in 6 patients, including MRSA (4 patients),

Stenotrophomonas maltophilia (3 patients), and Pseudomonas aeruginoma (2

patients). All but one (patient 1) received various antimicrobial agents

(extended-spectrum cephalosporins, glycopeptides, ciprofloxacin, clindamycin, or

metronidazole) for seven to 14 days before acquisition of PDRABup. After

notification of positive culture for PDRABup, two patients (patients 2 and 5) did not

ciprofloxacin, or extended-spectrum cephalosporins, according to the susceptibility

results of the co-isolates. Five patients survived.

(i) Patient 1. A 68-year-old woman diagnosed with ischemic stroke and

underlying congestive heart failure and diabetes mellitus. Antibiotic with

amoxicillin-clavulanate was administered empirically for aspiration pneumonia. On

the 9th day of antibiotic treatment, PDRABup was recovered from a sputum specimen.

There was no co-isolate yielded from the first two samples (A1, A2). Urinary tract

infection with Klebsiella pneumoniae developed later and was successfully treated by

ceftazidime and then cefepime. PDRABup was repeatedly recovered from sputum at

an interval of one month.

(ii) Patient 3. A 76-year-old woman diagnosed with cerebellar hemorrhage and

respiratory failure with endotracheal intubation. Nosocomial pneumonia developed

and PDRABup was identified on the ninth day of hospitalization. Stenotrophomonas

maltophilia, Proteus mirabilis, Pseudomonas aeruginosa and Klebsiella pneumoniae

were co-isolate bacteria. After successful treatment with flomoxef, ciprofloxacin and

then meropenem, the pneumonia improved and the patient survived. But persistent

recovery of the PDRABup from sputum and urine was noted for an interval of 3

months.

hemorrhage. Ventilator-associated pneumonia, cardiogenic shock and acute renal

failure developed during hospitalization. PDRABup, P. aeruginosa were identified

from sputum and Candida species and P. aeruginosa from urine. The patient died of

severe sepsis and deteriorated multiple organ failure.

Bacter ial isolates. All these isolates were gram-negative bacilli,

oxidase-negative, non-hemolytic, lactose (10%)-negative and glucose-non-acidifiers.

Colonies on trypticase soy agar supplemented with 5% sheep’s blood were mucoid

and slightly pink. They were identified as A. lwoffii by the Vitek GNI system (97%

presumptive identification) and as A. baumannii/calcoaceticus complex by the API

20NE (76.8 to 83% identification) and as Acinetobacter species by the Phoenix

system (90% confidence value). A control stain, A. baumannii ATCC 19606, was 10%

lactose-positive and glucose-acidifier, and was identified as A.

baumannii/calcoaceticus complex by the Vitek GNI system (99% presumptive

identification) and the API 20NE (99% identification) and as Acinetobacter species

by the Phoenix system (92% confidence value) (table 2).

The 16S rRNA sequencing data of these isolates (650 nucleotides) were

identical and were comparable with identification of A. baumannii/calcoaceticus

complex or A. junii. The 16S-23S ITS PCR-RFLP and the following sequencing

baumannii. There was only one nucleotide difference of the 16S-23S ITS sequence

between our isolates and A. baumannii ATCC 19606. The percentage of match to

GenBank sequence U60279 (A. baumannii), U60280 (Acinetobacter ATCC19004,

genomospecies 3), U60281 (Acinetobacter ATCC17903, genomospecies 13), and

U60278 (A. calcoaceticus, genomospecies 1) was 99.0%, 95.7%, 95.7%, and 95.7%,

respectively.

Susceptibility testing. All these isolates were uniformly resistant to

ampicillin-sulbactam (MICs, 128->128 µg/ml), ceftazidime (MICs, 64->128 µg/ml),

piperacillin-tazobactam (MICs, 128->128 µg/ml), cefepime (MICs, 16-32 µg/ml),

aztreonam (MICs, 64-128 µg/ml), ciprofloxacin (MICs, 64-128 µg/ml), trovafloxacin

(MICs, 8-16 µg/ml), moxifloxacin (MICs, 4 µg/ml), garenoxacin (MICs, 8-32 µg/ml),

amikacin (MICs, >128 µg/ml), imipenem (MICs, 8-16 µg/ml), and meropenem (MICs,

16->128 µg/ml).

Among the five pairs of antimicrobial agents tested for synergy, only imipenem

and ampicillin-sulbactam showed an enhanced zone of inhibition between the two

disks for all 15 isolates (Fig. 1).

PFGE profiles. All the 15 isolates of the PDRABup had an identical PFGE

profile, which was different from those of the five PDRAB isolates (only one isolate

an interval of one month had identical PFGE profiles. The similar scenario was also

found among five isolates (four from sputum specimens and one from a urine sample)

DISCUSSION

Over the past two decades, Acinetobacter species has been increasingly

associated with nosocomial infection and colonization, particularly those occurred in

patients with respiratory tract infections who hospitalized in intensive care units.

Many nosocomial outbreaks due to A. baumannii, particularly carbapenem-resistant

isolates or PDRAB isolates have been reported. Infections caused by PDRAB pose a

clinical dilemma because of the lack of appropriate antimicrobial therapy at present.

According to our previous reports, the mortality rate in patients with bacteremia due

to PDRAB was as high as 60% (17). The most common sources of infection are

derived from respiratory tract, indwelling catheters, and wounds. Furthermore, a few

clones of PDRAB were implicated in nosocomial Acinetobacter infections at the

NTUH since 1998 and a major clone (clone 5) has widely circulated in different

settings of the hospital with widespread carbapenem use (14).

This report describes a PDRAB clone with a phenotype different from PDRAB

clones found at the NTUH before and characterizes a nosocomial outbreak due to

these organisms during a 3-month period. Our results show three important facets.

First, although glucose-nonoxidizing A. baumannii accounts for 5% of all clinical A.

baumannii isolates (3, 25), isolates of A. baumannii (including PDRAB isolates) with

of problems in the routine clinical microbiology laboratories in speciation of

Acinetobacter species, a phenotypic scheme for identification of genomospecies 1 to

12 has previously been described (3), however, by using this system, discrepancies

with identifications by DNA-DNA hybridization, 16S rRNA sequencing, and

16S-23S ITS PCR-RFLP and sequencing have been found (6, 10, 16). In the present

study, the biochemical profiles of these asaccharolytic isolates (glucose-, lactose-,

xylose-, and mannitol-non-oxidizing strains) of Acinetobacter species generated by

the three commercial biochemical identification kits could not categorize to any

genomospecies of Acinetobacter (3, 7, 25). Although the 16S rRNA sequencing data

only suggested the identification of A. baumannii/calcoaceticus complex or A. junii,

the 16S-23S ITS PCR-RFLP and the following sequencing analysis confirmed the

identification of A. baumannii.

Second, this PDRABup had a PFGE profile different from those of 10 clones of

PDRAB recognized in our previous study indicating that these PDRABup isolates

belonged to a newly emerging clone. As seen in other PDRAB and other

non-fermentative gram-negative bacteria illustrated before, this clone isolates could

also have long-term persistence (infection or colonization) in humans for weeks and

months.

carbapenems, fluoroquinolones, aminoglycosides, and ampicillin-sulbactam. The high

MICs of these agents exceeded the levels achievable in plasma and tissue suggesting

their limited role as treatment regimen. Recent studies have demonstrated that

sulbactam alone at higher doses or in combinations with other agents provide efficacy

against nosocomial infections caused by multiresistant A. baumannii. Although

synergy was detected by disk method for combination of only imipenem plus

ampicillin-sulbactam, more studies including time-kill and in vivo animal studies

should be performed to establish the treatment options.

The environmental source and mode of spread of these PDRABup is obscure.

Previous studies have clearly demonstrated that air humidifiers, intravascular

institution fluids, feeding syringe, ventilator monitor board, resuscitation bags, hands

of medical staff, and intestinal flora have been found to be reservoirs and associated

with nosocomial outbreaks (14). During the period of this outbreak (from December

2002 to February 2003), several small clustering of PDRAB still occurred in intensive

care units and environmental surveillance (samples from air humidifiers, hands of

medical staff, mattresses, stock solution, sinks, taps, and portable X-ray machine)

failed to find this organisms. Fortunately, this clone circulated in the hospital for three

months and disappeared spontaneously, however, the classic PDRAB isolates was still

In this study, the majority of patients acquired PDRABup infections or

colonizations one week to more than one months after hospitalization, particularly

those received mechanical ventilation therapy. Clinically, it is difficult to determine

the pathogenic role of this organism because of the poor underlying medical condition

of the patients, the polymicrobial entity in specimens of infected sites and absence of

concurrent bacteremia due to this organism. In the two patients (patients 6 and 7) who

died, two well-known pathogens (P. aeruginosa and MRSA, respectively) were also

identified.

In summary, we report a nosocomial outbreak due to a novel PDRAB clone

occurred in seven patients at the hospital during a 3-month period. Because of the lack

of sufficient phenotypic discriminating criteria for identification of Acinetobacter

species, molecular methods should be conducted, particularly in isolates with unusual

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TABLE 2. Biochemical profiles of Acinetobacter baumannii ATCC19606 and the

clone of pandrug-resistant A. baumannii with unusual phenotype (PDRABup)

Biochemical profiles

A. baumannii The clone

Reaction ATCC19606 (PDRABup)

Growth at 37oC + + 41oC + + Acid from Glucose + − Lactose + − Sucrose − − Galactose − − Fructose − − Mannitol − − α-ketoglutaric acid + + Tiglic acid + + Utilization of Acetate + +

Citrate + + Malonate + + Glycine + + Leucine + + Arginine dihydrolase + − Orinithine decarboxylase − − Urea − − Esculin hydrolysis − −

FIG. 1. An enhanced zone of inhibition between imipenem and ampicillin-sulbactam

FIG. 2. Profiles obtained by PFGE for A. baumannii after digestion with SfiI. Lane M,

molecular size marker; lanes 1 to 11, pandrug-resistant A.baumannii with unusual

phenotype (PDRABup) isolates of A1. A3, A4, B, C1, C4, C5, D, E, F, and G (see

table 1 for designation of isolates); and lane 12, a PDRAB isolate belonging to clone 5,

Table 1. Clinical characteristics of seven patients with positive cultures for pan-drug resistantAcinetobacter baumannii with unusual phenotype who were treated at the

National Taiwan University Hospital

Isolation of A. baumannii Patient no. Sex/Age (yr) Underlying diseasesa Clinical syndrome

Site designation Date

(d/mo/yr) Co-isolates 1 F/68 Ischemic stroke, RHD, CHF, DM, UTI, pneumonia Fever Sputum Sputum Sputum Sputum A1 2/12/2002 A2 18/12/2002 A3 23/12/2002 A4 2/1/2003 A. baumannii, S. malsophilia, MRSA 2 M/81 Old stroke, BPH, recurrent UTI

Fever Urine B 5/12/2002 None

3 F/76 Cerebellar hemorrhage, post tracheostomy, hypertension, pneumonia, UTI Fever Sputum Sputum Sputum Sputum urine C1 9/12/2002 C2 30/12/2002 C3 10/1/2002 C4 20/1/2003 C5 27/1/2003 S. maltophilia, P. mirabilis, P. aeruginosa, K. pneumoniae, C. albicans 4 F/14 Encephalitis, status epilepticus, respiratory failure, UTI Fever Sputum, pressure sore D 12/12/2002 MRSA

respiratory failure, UTI sore 5 M/60 Old stroke, DM, CHF, renal insufficiency, hypoxic encephalopathy, UTI Shock (no fever) Wound (amputati on site) E 21/1/2003 MRSA 6 F/90 Head injury, intracranial hemorrhage, CHF, cardiogenic shock, cellulites, acute renal failure, pneumonia

Fever Sputum F 2/2/2003 P. aeruginosa

7 M/65 Chronic renal

failure, UTI,

cholangiocarcinom

a with obstructive

jaundice and BTI,

operated Fever, shock Sputum, urine, PTCD G 20/2/2003 MRSA, C. menigosepticum, S. maltophilia

a with obstructive

jaundice and BTI,

operated

a

BPH, benign prostatic hypertrophy; BTI, biliary tract infection; CHF, congestive

heart failure; DM, diabetes mellitus; MRSA, methicillin-resistant Staphylococcus

aureus; PTCD, percutaneous transhepatic cholangial drainage; RHD, rheumatic heart

disease; UTI, urinary tract infection.

1 2 3 4 5 6 7 8 9 10 11 12

Isolate _{A1 A3 A4 B C1 C4 C5 D E F G H}