Pseudomonas aeruginosa infection among cystic fibrosis and ICU patients in the referral children medical hospital in Tehran, Iran.

INTRODUCTION: Pseudomonas aeruginosa is one of the important causes of hospital-acquired infections in Intensive Care Unit (ICU) and considered as a major determinant of morbidity and mortality in patients affected by cystic fibrosis (CF). The aim of this study was to investigate clonal diversity among randomly picked P. aeruginosa isolates of CF and the other hospitalized patients in ICU.
METHODS: Cultivation, identification, and antimicrobial susceptibility testing of P. aeruginosa isolates were performed using standard techniques. The genetic similarity of the strains was investigated by amplification of the Enterobacterial Repetitive Intergenic Consensus-polymerase chain reaction (ERIC-PCR) sequence. RESULTS AND DISCUSSION: Among 49 isolates, sixteen were isolated from 11 patients affected by CF and 33 came from an epidemiological investigation of 25 P. aeruginosa infected patients of ICU. Five clusters were generated for all isolates analyzed through ERIC-PCR genotyping. Two major clusters (B and C) were discovered in P. aeruginosa isolates of ICU and CF patients during the whole period of this study. Fifteen unique antibiogram patterns obtained from all isolates and multi-resistant P. aeruginosa (MRPA) were identified in 23 isolates (47%). MRPA isolates were detected in all clusters (except A) while pan-resistant isolates were recovered only in cluster C. The high prevalence of related or identical isolates in CF and non-CF patients can be due to transmission of particular dominant clones in ICU ward. Therefore, enhanced infection-control may become necessary to prevent further spread of clonal strains.

Introduction

Pseudomonas aeruginosa is one of the important causes of hospital-acquired infections in intensive care unit (ICU) and considered as a major determinant of morbidity and mortality in patients affected by cystic fibrosis (CF) [1-3].

Surveillance of nosocomial P. aeruginosa infections has revealed trends of increasing antimicrobial resistance [4]. The emergence of transmissible P. aeruginosa strains at hospital and its spread to other P. aeruginosa-negative patients raise concerns especially in ICU settings, where the number of antibiotic agents with good activity is limited [5, 6]. Several studies describe the patient- to-patient transmission of this bacteria from centers related with CF patients [7, 8].

At our hospital, CF patients are cared in gastroenterology ward and P. aeruginosa-positive and P. aeruginosa- negative patients are not separated from each other in this ward. In addition, these patients move to other wards especially ICU during their hospitalization. The aim of this study was to investigate epidemiology of antimicrobial resistance and clonal diversity among randomly picked P. aeruginosa isolates of CF and the other hospitalized patients in ICU.

Material and methods

Between January and December 2010, randomly picked P. aeruginosa isolates of infected patient in ICU and patients affected by CF were collected from the tertiary referral Children Medical Hospital in Tehran, Iran. The patients investigated in this study were all prone to P. aeruginosa infection. An infected patient was defined as a patient with clinical symptoms of infection, and from whom a clinical culture yielded a dominant growth of P.aeruginosa. Clinical information on P. aeruginosa patient isolates was collected from medical records. Information included age, sex, length of hospital stay, history of transfer from another wards, clinical outcome and microbiological data.

A number of CF patients had more than one isolates and some of them were isolated while they were transferred to the ICU. In addition several cultures were obtained from some patients as long as they stayed in the ICU. Cultivation, identification, and antimicrobial susceptibility testing of P. aeruginosa isolates were performed using standard techniques [9].

Multi-resistant P. aeruginosa (MRPA) was defined as strains resistant to ≥ 3 of the following classes of antibiotics: antipseudomonal penicillins, antipseudomonal oxyimino- b-lactams, fluoroquinolones, aminoglycosides, and carbapenems [10].

The genetic similarity of the strains was investigated by amplification of the Enterobacterial Repetitive Intergenic Consensus-polymerase chain reaction (ERIC-PCR). sequence [11]. Comparison of banding patterns was performed using Gelcompar II, version 6.5 (Applied Maths, Sint-Matens-latem, Belgium). Cluster analysis was accomplished with the unweighted pair group method using average linkages (UPGMA). ERIC-PCR was performed for P. aeruginosa isolates and relatedness among genetic clones was defined as 80% similarity as belonging to one clone.

Results

During one year, 36 patients aged 3.5 to 192 month (average 33.4) were entered to this study. Table I illustrated the patient details, origin and date of isolation, outcome, ERIC-PCR and antibiogram pattern of all isolates.

Tab. I.

Patient details, origin and date of isolation, outcome and microbiological data of all the P. aeruginosa isolates.

Patient*	Isolates	Age (month)	Gender	Ward	Type of specimen	Length of hospital stay(Day)	Time of isolation after hospitalization(Day)	Outcome	ERIC-PCR pattern	Antibiogram pattern
1	a	11	M	PICU	Trachea tube	21	4	Death	D	12
2	a	12	M	PICU	Wound	30	6	Discharge	B	3
3	a	12	F	PICU	Ear secretion	58	5	Discharge	C	15
4	a	7	M	PICU	Trachea tube	34	20	Discharge	E	11
5	a	2.5	F	PICU	Urine	29	2	Discharge	C	15
6	a	10	F	PICU	Urine	30	21	Death	C	15
7	a	6	F	PICU	Wound	11	10	Death	A	6
8	a	3	M	PICU	Urine	21	5	Death	B	7
9	a	11	F	PICU	Trachea tube	5	2	Discharge	B	8
10	a	3	F	PICU	Wound	22	12	Death	B	7
	b				Eye		22		B	4
11	a	7	F	PICU	Trachea tube	15	5	Discharge	B	4
12	a	120	M	PICU	Urine	13	2	Discharge	B	3
13	a	2	M	PICU	Trachea tube	5	3	Death	B	11
14	a	9	M	PICU	Trachea tube	39	4	Discharge	B	4
	b				Wound		17		B	3
15	a	108	F	PICU	Trachea tube	54	2	Discharge	B	2
16	a	24	M	PICU	Trachea tube	4	3	Discharge	A	7
17	a	4	F	PICU	Blood	28	4		E	10
	b				Alveolar aspirate		14	Discharge	A	3
	c				Sputum		8		A	3
	d				Alveolar aspirate		8		C	12
18	a	8	M	PICU	Trachea tube	18	2	Discharge	C	2
	b				Urine		14		C	1
19	a	36	F	PICU	Wound	40	3	Discharge	B	7
	b		F				5		C	14
	c		F				20		C	12
20	a	4	M	PICU	Urine	25	1	Discharge	C	15
21	a	31	F	PICU	Urine	26	5	Discharge	B	3
22	a	36	M	PICU	Blood	58	1	Discharge	C	15
23	a	120	M	PICU	Trachea tube	19	4	Discharge	B	3
24	a	24	M	PICU	Pharynx	15	6	Discharge	C	14
25	a	3	F	PICU	Wound	51	3	Death	C	15
26	a	12	F	PICU	Wound	14	14	Death	C	15
27	a	192	F	Gastroenterology	Alveolar aspirate	56	4	Death	B	6
	b			Gastroenterology	Sputum		40		B	7
	c			Gastroenterology	Sputum		46		B	6
28	a	72	M	PICU	Trachea tube	12	2	Death	B	5
	b			PICU	Tracheal aspirate		7		B	13
	c			PICU	Trachea tube		11		B	11
29	a	5	F	PICU	Sputum	10	1	Dischage	C	15
30	a	45	M	Gastroenterology	Sputum	14	2	Dischage	B	8
31	a	120	M	Gastroenterology	Sputum	12	2	Death	B	7
32	a	10	M	PICU	Wound	10	9	Death	C	15
33	a	5	F	Gastroenterology	Urine	11	1	Dischage	B	9
34	a	7	M	Gastroenterology	Urine	36	2	Death	C	15
35	a	72	M	PICU	Trachea tube	7	2	Death	B	3
	b			Gastroenterology	Sputum	7	7		C	15
36	a	48	F	Gastroenterology	Sputum		1	Dischage	B	10

Isolates number of 26 to 36 isolated from CF patients. PICU: Pediatric Intensive Care Unit

Among 49 isolates, sixteen were isolated from 11 patients affected by CF and 33 came from an epidemiological investigation of 25 P. aeruginosa strains isolated from infected patients of ICU.

P. aeruginosa isolates were recovered from trachea tube (26%), urine (18%), wound (16%), sputum (18%), alveolar aspirate (8%), blood (4%), tracheal aspirate (2%), ear secretion (2%), and eye (2%).

Average length of hospital stay in all patients was 24 days. Of the 36 patients, 14 died during the study (case fatality rate, 39%); whereas half of them were from CF patients (case fatality rate, 64%). MRPA were identified in 23 isolates (47%) and consistently detected during the study period. Among all isolates, 11 considered as panresistant isolates that 5 of them belonged to CF patients. Five clusters were generated for all isolates analyzed through ERIC-PCR genotyping, which were designated alphabetically from A to D (Fig. 1). Cluster B and C comprised both ICU and CF isolates while cluster A, D and E had only isolates of ICU ward. Eleven isolates of cluster B were identical whereas cluster C comprised of only 3 identical isolates.

Fig. 1.

Dendrogram of genotype analysis derived from 49 P. aeruginosa isolates. The scale at the top represents the genetic distance between the isolates..

MRPA isolates were detected in all clusters (except A) while pan-resistant isolates were recovered only in cluster C.

Cluster B and C was seen during the study period (4 seasons) (Fig. 2). Cluster A was introduced during summer and remained present during the rest of the study. During the autumn, one patient appeared in cluster D and E. However, cluster D emerged in the third season, detection of this cluster during the winter did not occur.

Fig. 2.

Distribution of different clusters during 2010.

In our study, 15 unique antibiogram patterns obtained from all isolates (Tab. II). Most of the isolates were resistant to antipseudomonal oxyimino-b-lactams. The highest percentage of susceptibility was seen in aminoglycosides (71%) followed by piperacillin-tazobactam (67%), imipenem (63%), ciprofloxacin (59%), and meropenem (55%). Different antibiogram patterns were displayed in a number of isolates even with ≥ 95% similarity.

Tab. II.

Antibiogram patterns of 49 P. aeruginosa isolates.

Pattern number	Isolates*	CF	FEP	CP	MEM	CAZ	PTZ	IMP	GM	AM
1	18b	S	S	S	S	S	S	S	S	S
2	15,18a	S	S	S	S	R	S	S	S	S
3	2a,12,14b,17b,17c,21,23,35a	R	S	S	S	S	S	S	S	S
4	10b,11,14a	R	S	S	S	R	S	S	S	S
5	28a	S	S	S	R	S	S	S	R	S
6	7,27a,27c	R	R	S	S	S	S	S	S	S
7	8,10a,16,19a,27b,31	R	R	S	S	R	S	S	S	S
8	9,30	R	R	S	R	R	S	S	S	S
9	33	R	R	R	R	R	S	S	S	S
10	17a,36	R	R	S	R	R	R	S	S	S
11	2c,10b,28c	R	R	R	S	R	S	R	S	S
12	1,17d,19c	R	R	R	R	R	R	S	S	S
13	28b	R	R	S	S	R	S	R	R	R
14	19b,24	R	R	R	R	R	S	R	R	R
15	2b,5,6,20,22,25,26,29,32,34,35b	R	R	R	R	R	R	R	R	R

CF, Cephalothin; FEP, Cefepime; CP, Ciprofloxacin; MEM, Meropenem; CAZ, Ceftazidime; PTZ, Piperacillin-tazobactam; IMP, Imipenem; GM, Gentamycin; AM, Amikacin

Isolate number of 26 to 36 belongs to CF patients

Discussion

Our hospital is a tertiary referral center in which the ICU setting includes mixed patients such as CF patients and transmission of P. aeruginosa strains between CF and the other hospitalized patients in ICU is probable,Therefore genotyping of P. aeruginosa isolates seems to be essential to clarify our hospital epidemiology. The spread of infection from patients with CF is a definite risk in children's wards especially in ICU [12, 13]. In this study, molecular typing suggests cross-transmission between CF and non- CF patients. According to our results, infections were predominantly caused by strains with cluster B and C. presence of these patterns during the whole period of this study suggesting that these clones are adapted to our hospital. Cluster B was identified in 25 strains (51% of all typed isolates) and highly appeared in the first 3 months of 2010 and circulated until end of the study.

Our analysis demonstrated that CF isolates are genotypically closely related to non-CF isolates. The two major clusters (B and C) in our study were discovered in both patient populations that suggest probable occurrence of cross-infection between these patients.

Studies about P. aeruginosa cross-infection demonstrated controversy evidence of clonal spread in CF centers. Some studies have revealed convincing evidence of clonal spread at CF holiday camps or centers [14-18]. Another study at the Vancouver CF Centre did not represent any evidence of significant cross-infection with P. aeruginosa [19].

Emergence of MDR expect to become more prevalent in many hospitals [20, 21]. There is no doubt that cross-transmission plays an important epidemiological role in MRPA isolates [22, 23], so prevention of the acquisition of these isolates are essential due to limited therapeutic options and increased mortality [24]. On the other hand, prevalence of strains with resistance to all antimicrobial agents is a major risk for hospitalized patients especially in CF patients because of rare P. aeruginosa eradication and subsequently occurrence of chronic infection [25, 26].

P. aeruginosa has capacity to develop resistance to essentially antimicrobial agents [27] and may require treatment with less commonly used antibacterial agents such as colistin [4].

Our results support other studies [28] that mentioned aminoglycosides are clinically effective against P. aeruginosa isolates when administrated intravenously or by nebulization.

Finally, in our study the high prevalence of related or identical isolates in CF and non–CF patients can be due to transmission of particular dominant clones in ICU ward. This suggests cross-infection can occur between CF and non-CF patients. Therefore, enhanced infection-control including strict segregation policies, basic hygiene measures and continued microbiological surveillance may become necessary to prevent further spread of clonal strains.