Epidemiology and Antimicrobial Sensitivities of 536 Multi-Drug-Resistant Gram-Negative Bacilli Isolated from Patients Treated on Surgical Wards

Abstract

Background:

In this era of increasing antimicrobial resistance, infections caused by multi-drug-resistant (MDR) gram-negative bacilli (GNB) are becoming more common and pose a challenge to all clinicians, including surgeons.

Methods:

We evaluated the epidemiology and antimicrobial sensitivities of GNB isolated from patients treated on surgical wards at the University Hospital of Heraklion, Crete, Greece, from 2004 to 2009. The MDR isolates were defined according to an international expert proposal supported by the U.S. Centers for Disease Control and Prevention and the European Centre for Disease Prevention and Control.

Results:

A total of 1,153 GNB were isolated; 536 (46.5%) were MDR. The most common isolates were Escherichia coli (312 [27.8%]; MDR rate 50.2%), Pseudomonas aeruginosa (298 [25.8%]; MDR rate 39.6%), Acinetobacter baumannii (137 [11.9%]; MDR rate 83.9%), and Klebsiella pneumoniae (112 [9.7%]; MDR rate 44.6%). Most pathogens were isolated from patients hospitalized in the Departments of Surgical Oncology (32.3%), Orthopedic and Trauma Surgery (31.8%), General Surgery (18.1%), and Pediatric Surgery (15.5%). The clinical specimens comprised pus (45.1%), normally sterile fluids (22.5%), urine (16.8%), blood (6.3%), and other body fluids. Most effective in vitro against all MDR pathogens were colistin (83%), meropenem (57%), and imipenem-cilastatin (56%). The MDR P. aeruginosa was susceptible most often to colistin (94%) and aminoglycosides (tobramycin 56%, amikacin 55%), MDR A. baumannii only to colistin (94%), and MDR K. pneumoniae to meropenem (92%) and aminoglycosides (amikacin 76%, gentamicin 74%).

Conclusion:

In a region with a high prevalence of antibiotic resistance, almost one-half of GNB isolated from surgical patients were MDR. Surgeons may consider these developments to guide empiric antibiotic therapy for infections caused by gram-negative pathogens.

Infection is a troublesome complication in surgical patients. The most common type of infection in this population is surgical site infection (SSI); SSIs are the third most common hospital-acquired infections overall [1]. Other major sites of infection in surgical patients are the urinary tract, respiratory tract, and blood stream; these infections may be associated with the use of indwelling urinary catheters, mechanical ventilators, or intravenous catheters [2].

The overuse and misuse of antibiotics has led bacterial species to develop various resistance mechanisms in order to survive [3]. Thus, multi-drug-resistant (MDR) bacteria now are an increased clinical concern [4]. Gram-positive cocci appear as the most frequent causative pathogens in SSIs; fortunately, several novel agents have been approved in recent years, namely linezolid [5], daptomycin [6], and telavancin. In contrast, novel agents targeting MDR gram-negative bacteria are scarce [7]. Limited additional therapeutic options against those pathogens have taken the form of revival of older antibiotics such as colistin [8] and fosfomycin [9].

The prevalence of gram-negative bacilli (GNB) among surgical patients appears to be increasing [2]; and, in cases of MDR pathogens, surgeons may have limited therapeutic options. In this context, we documented the epidemiologic characteristics and antimicrobial sensitivity patterns of GNB isolated from surgical patients at a tertiary-care university hospital in a region with high antibiotic resistance rates.

Patients and Methods

Study design and setting

All clinical isolates of GNB collected over a six-year period (2004–2009) by the microbiology laboratory of the University Hospital of Heraklion (Heraklion, Crete, Greece), for which sensitivity testing to isepamicin had been performed, were tested.* This hospital is a 680-bed tertiary-care center that serves a population of approximately 650,000 individuals. Only pathogens from patients treated in the General Surgery, Orthopedic and Trauma Surgery, Thoracic and Cardiac Surgery, Surgical Oncology, and Pediatric Surgery departments were eligible for analysis; no isolates from the intensive care units (ICUs) were included. All isolates originating from clinical specimens were evaluated, as were all isolates of different species originating from the same clinical specimen. When multiple isolates of the same species were identified from the same patient more than once in six months, only the first was included.

Routine laboratory methods were used for specimen processing and culture. The identification of bacterial species was performed by standard biochemical methods, the API system (BioMérieux, Marcy l’ Etoile, France), and the Vitek2 automated system BioMérieux). The Vitek2 automated system also was used for the antimicrobial sensitivity testing. The Clinical and Laboratory Standards Institute (CLSI) breakpoints (M100-S20) were applied to interpret susceptibility results for all agents [10]. Isolates were not tested against fosfomycin or newer antibiotics such as tigecycline and ceftaroline.

Definitions and data analysis

Multi-drug-resistant isolates were defined according to an international expert proposal for interim standard definitions for acquired resistance, supported by the European Centre for Disease Prevention and Control (ECDC, Stockholm, Sweden) and the U.S. Centers for Disease Control and Prevention (CDC, Atlanta, GA) [13]. According to this definition, to be considered MDR, an isolate should have acquired resistance (non-sensitivity) to more than one agent in three or more antimicrobial classes [13].

Dichotomous variables were compared using chi-square tests. Statistical significance was determined at 0.05. All analyses were performed with SPSS version 17.0 software (SPSS Inc., Chicago, IL).

Results

Epidemiology of the clinical isolates

During the study period, 1,153 GNB were isolated from surgical patients. Of these, 46.5% (536) were MDR. An overview of the pathogens is presented in Table 1. The most common were Escherichia coli (321; 27.8%), Pseudomonas aeruginosa (298; 25.8%), Acinetobacter baumannii (137; 11.9%), and Klebsiella pneumoniae (112; 9.7%). Similarly, the most common MDR pathogens were E. coli (161/536; 30%), P. aeruginosa (118; 22%), A. baumannii (115; 21.5%), and K. pneumoniae (50; 9.3%).

Table 1.

Overview of 1,153 Gram-Negative Bacilli, Including the 536 Multi-Drug-Resistant (MDR) Isolates

	N₁ (N₁/N [%]) ^a	MDR (N₂, [N₂/N₁] [%])
Enterobacteriaceae (n=682; 280 [41%] MDR)
Escherichia coli	321 (27.8)	161 (50.2)
Klebsiella pneumoniae	112 ( 9.7)	50 (44.6)
Klebsiella oxytoca	28 ( 2.4)	12 (42.9)
Enterobacter spp.	90 ( 7.8)	28 (31.1)
Proteus mirabilis	55 ( 4.8)	8 (14.5)
Proteus spp. (not P. mirabilis)	15 ( 1.3)	0
Citrobacter spp.	28 ( 2.4)	15 (53.6)
Serratia marcescens	10 ( 0.9)	2 (20)
Morganella morganii	10 ( 0.9)	0
Salmonella spp.	5 ( 0.4)	0
Other enterobacteriaceae^b	8 ( 0.7)	4 (50)
Non-fermentative gram-negative bacilli (n=471; 256 [54%] MDR)
Pseudomonas aeruginosa	298 (25.8)	118 (39.6)
Pseudomonas spp. (not P. aeruginosa)	9 ( 0.8)	0
Acinetobacter baumannii	137 (11.9)	115 (83.9)
Acinetobacter spp. (not A. baumannii)	5 ( 0.4)	4 (80.0)
Alcaligenes spp.	11 ( 1.0)	9 (81.8)
Aeromonas spp.	5 ( 0.4)	4 (80.0)
Bordetella bronchiseptica	5 ( 0.4)	5 (100)
Pasteurella multocida	1 ( 0.1)	1 (100)

N=total number of isolates; N₁=number of particular bacterial species; N₂=number of MDR isolates of that species.

Including Providencia spp., Serratia spp., and Escherichia spp.

The origins of the tested isolates are presented in Table 2. Most pathogens were isolated from the Departments of Surgical Oncology (372; 32.3%) and Orthopedic and Trauma Surgery (367; 31.8%). The remaining pathogens were isolated from the Departments of General Surgery (209; 18.1%), Pediatric Surgery (179; 15.5%), and Thoracic and Cardiac Surgery (26; 2.3%). Similar patterns applied to the MDR isolates. Regarding specific pathogens, A. baumannii was isolated more commonly than other GNB from Orthopedic and Trauma Surgery patients (59.1% vs. 28.1%; p<0.001), K. pneumoniae from General Surgery patients (27.7% vs. 17.1%; p=0.006), and E. coli from Pediatric Surgery patients (27.4% vs. 36.5%; p<0.001).

Table 2.

Origin of the 1,153 Isolates a

Department	All isolates	Pseudomonas aeruginosa	Acinetobacter baumannii	Klebsiella pneumoniae	Escherichia coli
General Surgery	209	47 (22.5)	24 (11.5)	31 (14.8)	61 (29.2)
	104	21 (20.2)	22 (21.2)	15 (14.4)	32 (30.8)
Thoracic and Cardiac Surgery	26	6 (23.1)	7 (26.9)	4 (15.4)	3 (11.5)
	13	1 ( 7.7)	7 (53.8)	2 (15.4)	1 ( 7.7)
Orthopedic and Trauma Surgery	367	106 (28.9)	81 (22.1)	28 (7.6)	63 (17.2)
	182	44 (24.2)	65 (35.7)	15 (8.2)	28 (15.4)
Surgical Oncology	372	92 (24.7)	21 (5.6)	41 (11.0)	106 (28.5)
	169	38 (22.5)	20 (11.8)	17 (10.1)	59 (34.9)
Pediatric Surgery	179	47 (26.3)	4 ( 2.2)	8 ( 4.5)	88 (49.2)
	68	14 (20.6)	1 ( 1.5)	1 ( 1.5)	41 (60.3)

Number per department (%); number of MDR organisms (%).

The clinical specimens from which the isolates derived are listed in Table 3. Most pathogens were isolated from pus (520/1153; 45.1%), normally sterile fluid (including peritoneal fluid, bile, pleural fluid, pancreatic fluid, synovial fluid, cerebrospinal fluid, and otherwise non-specified fluid; 260; 22.5%), urine (194; 16.8%), and blood (including intravenous catheter tip; 73; 6.3%) samples. The remaining clinical specimens were respiratory tract secretions (45; 3.9%), tissue samples (48; 4.2%), and other materials (including genital, stool, and bone marrow aspirate specimens; 13; 1.1%). Similar patterns applied to the MDR isolates.

Table 3.

Source of Pathogens a

Specimen	All isolates	Pseudomonas aeruginosa	Acinetobacter baumannii	Klebsiella pneumoniae	Escherichia coli
Pus^b	520	143 (27.5)	59 (11.3)	38 (7.3)	126 (24.2)
	228	51 (22.4)	49 (21.5)	15 (6.6)	67 (29.4)
Blood^c	73	12 (16.4)	18 (24.7)	13 (17.8)	7 (9.6)
	46	7 (15.2)	15 (32.6)	7 (15.2)	4 (8.7)
Urine	194	33 (17.0)	15 (7.7)	23 (11.9)	95 (49.0)
	93	16 (17.2)	13 (14.0)	13 (14.0)	47 (50.5)
Normally sterile fluid^d	260	71 (27.3)	21 (8.1)	30 (11.5)	83 (31.9)
	120	26 (21.7)	19 (15.8)	13 (10.8)	41 (34.2)
Peritoneal fluid	74	18 (24.3)	7 (9.5)	5 (6.8)	22 (29.7)
	29	8 (27.6)	5 (17.2)	2 (6.9)	9 (31.0)
Respiratory tract specimens	45	21 (46.7)	14 (31.1)	3 (6.7)	3 (6.7)
	28	10 (35.7)	12 (42.9)	2 (7.1)	1 (3.6)
Tissue	48	16 (33.3)	10 (20.8)	3 (6.3)	5 (10.4)
	19	7 (36.8)	7 (36.8)	0	1 (5.3)
Other specimens^e	13	2 (15.4)	0	2 (15.4)	2 (15.4)
	2	1 (50.0)	0	0	0

Number per type of specimen (%); number of MDR organisms (%).

Including abscess (18 specimens).

Including intravenous catheter tips (41 specimens).

Peritoneal fluid (74 specimens), bile (11), pleural fluid (7), pancreatic fluid (2), synovial fluid (1), cerebrospinal fluid (1), and otherwise non-specified fluid (160).

Genital (8), stool (4), and bone marrow aspirate (1) specimens.

Regarding specific pathogens, P. aeruginosa was isolated more frequently than other GNB from respiratory tract specimens (7% vs. 2.8%; p=0.001), A. baumannii from blood (13.1% vs. 5.4%; p<0.001) and respiratory tract specimens (10.2% vs. 3.1%; p<0.001), and K. pneumoniae from blood (11.8% vs. 5.2%; p=0.016). In addition, E. coli was isolated more frequently than other GNB from urine samples (29.6% vs. 11.9%; p<0.001) and Enterobacter spp. from peritoneal fluid (14.9% vs. 7.3%; p=0.02).

Antimicrobial sensitivity

The antimicrobial sensitivity of all MDR pathogens, as well as P. aeruginosa, A. baumannii, K. pneumoniae, and E. coli separately, are presented in Table 4. Crude data on non-MDR isolates also are provided. Colistin was the most effective antibiotic against all MDR GNB (82.5%), followed by the carbapenems (meropenem 56.9%, imipenem-cilastatin 56.3%). Regarding specific pathogens, MDR P. aeruginosa was most often susceptible to colistin (94.1%), tobramycin (55.9%), and amikacin (55.1%); and MDR A. baumannii was most often susceptible to colistin (93.9%), amikacin (20.9%), and carbapenems (18.3%). The MDR K. pneumoniae and E. coli were most often susceptible to meropenem (92% and 84.5%, respectively), imipenem-cilastatin (90% and 83.2%, respectively), and amikacin (76% and 74.2%, respectively).

Table 4.

Antimicrobial Susceptibility of Common Gram-Negative Bacillia,b

Antibiotic	All isolates (N_MDR=536; N_ALL=1153)	Pseudomonas aeruginosa (N_MDR=118; N_ALL=298)	Acinetobacter baumannii (N_MDR=115; N_ALL=137)	Klebsiella pneumoniae (N_MDR=50; N_ALL=112)	Escherichia coli (N_MDR=161; N_ALL=321)
Ampicillin	NA	NA	NA	NA	44.1 (54.2)
Amoxicillin-clavulanic acid	NA	NA	NA	34 (50)	76.4 (81.6)
Ticarcillin-clavulanic acid	29.1 (65.5)	1.7 (59.4)	6.1 (21.2)	58 (80.4)	55.9 (77.3)
Piperacillin-tazobactam	37.1 (70.2)	19.5 (66.8)	6.1 (21.2)	66 (84.8)	64 (81.6)
Cefalotin	NA	NA	NA	10 (47.3)	11.2 (45.2)
Cefoxitin	NA	NA	NA	34 (66.1)	39.8 (64.8)
Cefuroxime	NA	NA	NA	34 (67)	34.2 (64.2)
Ceftriaxone	NA	NA	5.2 (16.8)	66 (84.8)	63.4 (81.6)
Cefotaxime	NA	NA	5.2 (16.8)	68 (85.7)	64.6 (82.2)
Ceftazidime	39.6 (71.8)	28.8 (71.8)	6.1 (20.4)	66 (84.8)	64.6 (82.2)
Cefepime	40.7 (72.2)	28 (71.5)	3.5 (17.5)	72 (87.5)	68.3 (84.1)
Aztreonam	NA	13.6 (55.4)	NA	66 (84.8)	64 (81.9)
Imipenem-cilastatin	56.3 (79.7)	32.2 (73.2)	18.3 (31.4)	90 (95.5)	83.2 (91.6)
Meropenem	56.9 (80)	32.2 (73.2)	18.3 (31.4)	92 (96.4)	84.5 (92.2)
Tobramycin	50.4 (76.4)	55.9 (82.2)	12.2 (24.8)	72 (87.5)	67.7 (83.8)
Amikacin	55.8 (79)	55.1 (81.2)	20.9 (32.8)	76 (89.3)	74.2 (87.5)
Gentamicin	49.6 (75.6)	41.5 (75.2)	17.4 (29.2)	74 (88.4)	68.9 (84.1)
Netilmicin	46.5 (74.1)	40.7 (74.2)	8.7 (21.9)	70 (86.6)	69.6 (84.7)
Tetracycline	NA	NA	7.8 (19.7)	34 (59.8)	33.5 (61.7)
TMP/SMX	NA	NA	10.4 (24.8)	66 (81.3)	56.5 (76)
Ciprofloxacin	35.1 (69.2)	18.6 (67.8)	0.9 (14.6)	72 (86.6)	56.5 (77.3)
Colistin	82.5 (89.5)	94.1 (96.6)	93.9 (94.2)	74 (84.8)	68.9 (81)

Percent susceptibility rate of MDR isolates (% susceptibility rate of all isolates).

N_MDR=number of MDR isolates; N_ALL=number of all (MDR and non-MDR) isolates; NA=not appropriate (potentially intrinsically resistant).

TMP/SMX=trimethoprim/sulfamethoxazole.

Discussion

Our main finding is that a considerable proportion (46.5%) of GNB isolated from surgical ward patients in a tertiary-care hospital were resistant to one or more agents of three or more classes of antibiotics. The most effective antibiotics against all (including the MDR) isolates were colistin, the carbapenems (meropenem and imipenem-cilastatin), and certain aminoglycosides. Previous studies have highlighted the increasing antimicrobial resistance rates of bacteria implicated in surgical infections, especially GNB [14 –18].

Pseudomonas aeruginosa is a common pathogen in surgical patients [16,19,20]. It can cause hospital-acquired pneumonia, as well as intra-abdominal, soft tissue, blood stream, and surgical site infections [19,21]. Our findings regarding sensitivity to various antibiotics, including piperacillin-tazobactam (67%), anti-pseudomonal cephalosporins (72%), aminoglycosides (74%–82%), and carbapenems (73%), were similar to those documented in a worldwide surveillance study [22] published recently. Of interest, 40% (118/298) of P. aeruginosa strains were MDR. Alarmingly, only colistin (94%) and certain aminoglycosides (tobramycin 56%, amikacin 55%) exhibited considerable in vitro activity against MDR P. aeruginosa isolates (see Table 4).

A. baumannii infection is becoming more common in surgical patients, particularly those admitted to the ICU. The organism is considered a common cause of blood stream infection, hospital-acquired pneumonia, and SSI [23]. The isolates we evaluated appeared to have moderately higher resistance rates than those in the worldwide surveillance study [20]; piperacillin-tazobactam (21%), anti-pseudomonal cephalosporins (18%–20%), aminoglycosides (22%–42%), and carbapenems (31%) do not seem reasonable choices for the treatment of patients with such infections. Our findings regarding MDR strains (84% of the total) were somewhat worse; only colistin was effective in vitro (94%; Table 4). However, the isolates in our study were not tested against newer potentially effective agents such as tigecycline [24].

Klebsiella spp. (especially K. pneumoniae) are common in wound and blood stream infections in surgical patients [25]. Although generally an easy-to-treat enterobacterium, Klebsiella has a high capacity for acquired resistance [26]. The local strains of K. pneumoniae generally were susceptible to various antibiotics (piperacillin-tazobactam 85%, anti-pseudomonal ephalosporins 85%–88%, aminoglycosides 87%–98%, and carbapenems 96%), which are higher percentages than in the worldwide surveillance study [22]. Even against the MDR isolates (45% of the total), many antibiotics exhibited considerable activity in vitro, including the carbapenems (90%–92%), certain aminoglycosides (amikacin 76%, gentamicin 74%), colistin (74%), and ciprofloxacin (72%). Although one may be alarmed by the relatively low sensitivity of K. pneumoniae strains to colistin, various antibiotics retained their activity against those strains, as has been reported previously [27].

A recently published study suggested that adherence of surgeons to the current guidelines on antimicrobial prophylaxis is suboptimal worldwide [28], and Greece does not seem to be an exception [29]. As in other hospitals, it is possible that in the study institution, antimicrobial agents are overused on surgical wards, including those administered as prophylaxis [30]. A prospective audit of 2,420 operations found that antimicrobial prophylaxis was used for 76% of clean operations, with a median duration of three days, and combinations of antibiotics were common (38% of operations) [31].

The high antibiotic resistance rates among GNB isolated from surgical patients have important clinical implications. First, treatment options for infections caused by MDR GNB are limited; for instance, some isolates in our series were susceptible only to colistin. Treating such patients empirically with a beta-lactam, a fluoroquinolone, or even a carbapenem would be insufficient [32]. Second, the standard practice regarding antimicrobial prophylaxis in surgery may need to be reconsidered, at least in high-risk patients in areas with high antimicrobial resistance rates [33]. Last, surgeons may focus on the prevention of antibiotic resistance and on infection control. This could be implemented with strict adherence to the published guidelines regarding infection control and antimicrobial prophylaxis or with the evaluation of novel strategies such as antibiotic rotation in the ICU [34,35], selective digestive tract and oropharyngeal decontamination [36], or use of probiotics [37].

Our study has certain strengths and specific limitations. A major strength is that, to our knowledge, our sample is the largest published to date examining gram-negative MDR isolates implicated in surgical infections. Furthermore, all clinical specimens were isolated from surgical ward patients, thereby avoiding the effect of a different microbial epidemiology such as the ICU, which typically is characterized by higher antibiotic resistance rates [38]. However, it should be acknowledged that some of the patients may have had prior ICU admissions. In addition, the origin of the infections (nosocomial or community-acquired) could not be assessed, although the majority probably were nosocomial.

On the other hand, our study suffers the inherent limitations of single-center studies and retrospective analyses. Moreover, the isolates were not tested for clonality, and molecular methods in general were not used. Last, the data we analyzed referred to the majority (although not all) of the tested isolates during the study period; however, there was no systematic bias in the case selection.

In conclusion, almost one-half of the evaluated GNB isolated from surgical patients were MDR. This included resistance to advanced antibiotics (i.e., ciprofloxacin, carbapenems, third- and fourth-generation cephalosporins). Surgeons may keep these developments under consideration until future studies define the clinical implications of the phenomenon of increasing antibiotic resistance in surgical patients.

Footnotes

Author Disclosure Statement

The authors have no conflicts of interest to declare. No funding was provided for this research.

*

Part of the original data has been used in two studies evaluating the sensitivity of various pathogens to isepamicin [10,].

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