Increasing Incidence of Multidrug Resistance Among Cystic Fibrosis Respiratory Bacterial Isolates

Abstract

Pseudomonas aeruginosa and Staphylococcus aureus are common pathogens in cystic fibrosis (CF) patients with increasing multidrug resistance (MDR). This study characterized antimicrobial susceptibility trends among organisms isolated from the respiratory tract of CF patients. Microbiological culture and sensitivity results for all CF patients were collected from January 2010 through December 2014. Minimum inhibitory concentrations were obtained using Phoenix^® and Etest^® methods. Clinical and Laboratory Standards Institute guidelines were used to remove duplicate isolates and develop antimicrobial susceptibility reports. MDR was defined as resistance to one agent in three or more antibiotic classes or oxacillin resistance in S. aureus. Overall, 542 bacterial isolates from 376 cultures were analyzed for trends. P. aeruginosa (41%), S. aureus (40%), and Stenotrophomonas maltophilia (8%) were the most commonly isolated organisms. Multidrug-resistant organism isolation increased from 39% to 49% (r = 0.76, p = 0.13), while representing 47.6% of all isolates. Multidrug-resistant P. aeruginosa incidence increased each year from 26% to 43% (r = 0.89, p = 0.046), while P. aeruginosa isolation decreased from 47% to 38% over the study period (r = −0.93, p = 0.02). MRSA accounted for 62.6% of all S. aureus isolated, while overall multidrug-resistant S. aureus incidence was 73.1% in all cultures. MDR among common pathogens in CF continues to increase. Empiric therapy for CF exacerbations should be targeted to previous antimicrobial susceptibility, and P. aeruginosa and S. aureus should be empirically covered.

Background

P seudomonas aeruginosa and Staphylococcus aureus are the most commonly isolated organisms from the respiratory tract of cystic fibrosis (CF) patients. Incidence of S. aureus has steadily increased over the past decade, while P. aeruginosa incidence has declined.¹ Polymicrobial infection with P. aeruginosa and S. aureus is associated with worse respiratory function compared to infection with only S. aureus.^2,3 A recent study found that the prevalence of P. aeruginosa decreased and the prevalence of S. aureus infections increased from 2006 through 2012.⁴ In addition, increases in Stenotrophomonas maltophilia prevalence are reported.

Enterobacteriaceae are common pathogenic organisms in pneumonia among patients; however, these organisms are rarely implicated in CF respiratory infection and generally considered colonizers.⁵ Serratia marcescens is generally the most commonly isolated Enterobacteriaceae organism.⁵ Several cases of multidrug-resistant Enterobacteriaceae chronic infections have recently been reported.^6–8

The purpose of this study was to characterize antimicrobial susceptibility trends among organisms isolated from the respiratory tract of CF patients.

Materials and Methods

This study was conducted at the University of Kentucky HealthCare Chandler Medical Center (UKHC), a 689 bed tertiary academic medical center. UKHC is home to the NCI-designated Markey Cancer Center, Gill Heart Institute, in addition to being a certified adult and pediatric CF center. In 2014, the UKHC CF center had 1,118 adult and pediatric patient encounters with an average hospitalization length of stay of 26.4 days.

Respiratory culture and susceptibility data from all hospitalized CF patients were collected from January 2010 through December 2014 from the clinical microbiology laboratory. All CF specimens were cultured on selective OFPBL (BD BBL™) agar for Burkholderia cepacia complex in addition to standard media for all other organisms. OFPBL plates were incubated at 30°C and examined daily for 4 days. Antimicrobial susceptibilities were determined phenotypically using Etest^® methodology or an automated system (BD Phoenix II^®). Minimum inhibitory concentration values and Clinical and Laboratory Standards Institute (CLSI) interpretations for the year of collection were reported. The 2014 CLSI breakpoints were retrospectively applied to all years of data.⁹ Duplicate isolates within each calendar year were removed before analysis in accordance with CLSI recommendations for cumulative susceptibility reporting, with the first culture of each isolate per patient being retained in the data.¹⁰

Agents examined are those commonly tested in the clinical microbiology laboratory and reported. Antimicrobials reported for P. aeruginosa include the following: aztreonam, ceftazidime, cefepime, meropenem, piperacillin–tazobactam, amikacin, gentamicin, tobramycin, ciprofloxacin, and levofloxacin. Other carbapenems and polymyxins are tested only in situations where the isolate is known to be multidrug resistant, which would lead to skewed representation of susceptibility patterns in this study. In addition to the antimicrobials tested against Pseudomonas, Enterobacteriaceae are tested against the following: ampicillin, ampicillin-sulbactam, cefazolin, ertapenem, tetracycline, and trimethoprim–sulfamethoxazole. Third-generation cephalosporins are not commonly tested in Enterobacteriaceae due to the automated panels not containing these agents. Due to low isolate counts for individual species, Enterobacteriaceae antimicrobial susceptibility trends were analyzed as a group. S. maltophilia isolates are only routinely tested against trimethoprim/sulfamethoxazole, minocycline, and levofloxacin, with beta-lactam agents only being tested in isolates resistant to the routinely tested agents. Antimicrobial agents reported for S. aureus isolates include the following: oxacillin, penicillin, gentamicin, levofloxacin, clindamycin, erythromycin, minocycline, tetracycline, trimethoprim–sulfamethoxazole, linezolid, and vancomycin. Ceftaroline is not reported due to inconsistent testing (only available for 16 MRSA isolates).

Multidrug resistance (MDR) was defined as resistance to one agent in three or more antibiotic classes tested or oxacillin resistance in S. aureus.¹¹ In addition, we only included classes that were routinely tested against the organism. Extensively drug resistant (XDR) was defined as resistance to an agent in all but one class of antimicrobials tested. Pandrug resistance was defined as resistance to all classes of antimicrobials tested. A polymicrobial infection was defined as the presence of two or more microorganisms isolated from a single culture as noted by the clinical microbiology laboratory accession number. Trends in organism isolation and MDR prevalence were analyzed with Pearson's correlation coefficient with a prespecified alpha of 0.05.

Results

In total, 542 unique CF respiratory isolates from 376 cultures and 154 unique patients were obtained. There were 150 cultures with multiple organisms isolated from 82 patients, of which 61 (74%) patients were coinfected with P. aeruginosa and S. aureus. P. aeruginosa, S. aureus, and S. maltophilia were the most commonly isolated organisms. Table 1 describes all the isolates collected. Overall, MDR organism isolation increased from 39% in 2010 to 49% in 2014 (r = 0.76, p = 0.13), while representing 47.6% of all isolates.

Table 1.

Respiratory Bacterial Pathogens Isolated in Cystic Fibrosis Patients from January 2010 to December 2014, N (%)

Organism	2010	2011	2012	2013	2014	Organism total
Pseudomonas aeruginosa	42 (47.2)	47 (43.1)	41 (39.8)	45 (39.8)	49 (38.3)	224 (41.3)
S. aureus	36 (40.4)	45 (41.3)	44 (42.7)	46 (40.7)	48 (37.5)	219 (40.4)
S. maltophilia	6 (6.7)	6 (5.5)	8 (7.8)	11 (9.7)	14 (10.9)	45 (8.3)
E. coli	2 (2.2)	3 (2.8)	4 (3.9)	2 (1.8)	2 (1.6)	13 (2.4)
E. cloacae	1 (1.1)	2 (1.8)	2 (1.9)	2 (1.8)	3 (2.3)	10 (1.8)
E. faecium	—	—	1 (1.0)	1 (0.9)	5 (3.9)	7 (1.3)
E. faecalis	—	—	—	2 (1.8)	4 (3.1)	6 (1.1)
S. marcescens	—	3 (2.8)	1 (1.0)	1 (0.9)	1 (0.8)	6 (1.1)
S. pneumoniae	1 (1.1)	2 (1.8)	1 (1.0)	1 (0.9)	—	5 (0.9)
A. baumannii	—	—	1 (1.0)	1 (0.9)	—	2 (0.4)
K. pneumoniae	—	1 (0.9)	—	—	1 (0.8)	2 (0.4)
C. freundii	—	—	—	—	1 (0.8)	1 (0.2)
K. oxytoca	—	—	—	1 (0.9)	—	1 (0.2)
P. mirabilis	1 (1.1)	—	—	—	—	1 (0.2)
Total	89	109	103	113	128	542

There were 224 P. aeruginosa isolates (41% of all cultures) from 105 patients. Incidence of P. aeruginosa isolation decreased from 47% in 2010 to 38% of bacterial cultures in 2014 (r = −0.93, p = 0.02). Overall, 36% of P. aeruginosa isolates were MDR and 49 patients (46.7%) carried an MDR isolate; however, MDR-P. aeruginosa incidence increased each year from 26% to 43% (r = 0.89, p = 0.046). In addition, 30.8% of all P. aeruginosa isolated were characterized as mucoid strains (range 26.8–40%, r = 0.31, and p = 0.62). Overall susceptibilities for P. aeruginosa are problematic (Table 2), with no single agent possessing a percent susceptible of greater than 80%. Piperacillin–tazobactam and ceftazidime were the most likely agents to have a susceptible P. aeruginosa isolate (77% of isolates tested susceptible), followed by tobramycin (75%). In contrast, non-MDR P. aeruginosa remains reliably susceptible to most beta-lactams tested (ranging from 81% for cefepime to 98% for piperacillin–tazobactam, ceftazidime, and aztreonam); however, tobramycin (80% susceptible) is the only viable non-beta-lactam option for empiric systemic treatment of non-MDR P. aeruginosa from the present data. None of the agents tested against MDR-P. aeruginosa achieved a percent susceptible greater than 50% and tobramycin was the most active agent.

Table 2.

Antimicrobial Susceptibility for S. aureus, P. aeruginosa, Enterobacteriaceae, and S. maltophilia over Study Period in Cystic Fibrosis Isolates (% Susceptible)

		Beta-lactams											Aminoglycosides			Fluoroquinolones		Macrolides		Tetracyclines		Sulfa antibiotics	Antistaphylococcal agents
Organism	N	Ampicillin	Ampicillin–sulbactam	Aztreonam	Cefazolin	Ceftazidime	Cefepime	Ertapenem	Meropenem	Oxacillin	Penicillin	Piperacillin–tazobactam	Amikacin	Gentamicin	Tobramycin	Ciprofloxacin	Levofloxacin	Clindamycin	Erythromycin	Minocycline	Tetracycline	Trimethoprim–sulfamethoxazole	Linezolid	Vancomycin
Staphylococcus aureus	219									39	2			92			26	43	7	97	93	92	99	100
MRSA	137									0	0			94			5	27	1	97	91	94	99	100
MDR-MSSA	23									100	11			61			22	35	0	95	91	70	100	100
MSSA	59									100	6			98			76	81	22	100	98	97	100	100
Pseudomonas aeruginosa	224			73		77	52		70			77	47	55	75	44 (191)	49
MDR	80			33		44	6		32			44	20	30	49	17 (71)	20
Non-MDR	144			98		98	81		94			98	63	73	92	60 (120)	72
Enterobacteriaceae	34	9	30	97	8	97	100	100	100			82	97	73	74	75	74				44	64
MDR	14	0	0	93	0	93	100	100	100			57	100	64	64	43	43				7	18
Non-MDR	20	17	57	100	13	100	100	100	100			100	95	79	80	100	95				72	100
Stenotrophomonas maltophilia	45																62			100		91

Number in parentheses is number of isolates tested when significantly different than other agents.

Resistance patterns for P. aeruginosa are described in Table 3. Resistance in non-MDR P. aeruginosa is characterized primarily by aminoglycoside resistance with 38.2% of all isolates demonstrating resistance (65.5% of non-MDR P. aeruginosa displaying a resistance to at least one agent). Of the 28 XDR isolates, 22 (78.6%) were susceptible only to fluoroquinolone agents. Twelve isolates were resistant to every class tested and classified as pandrug resistant.

Table 3.

P. aeruginosa Drug Resistance Patterns

	No.	Percent
Pan susceptible	60	26.8
Resistant to one class	56	25.0
Aminoglycosides	39	69.6
Beta-lactam^a	10	17.9
Fluoroquinolones	7	12.5
Resistant to two classes	28	12.5
Aminoglycosides + beta-lactam	13	46.4
Fluoroquinolones + beta-lactam	8	28.6
Two beta-lactam classes	4	14.3
Aminoglycosides + fluoroquinolones	3	10.7
MDR	40	17.9
Aminoglycosides + fluoroquinolones + Beta-lactam	16	40.0
Aminoglycosides + two beta-lactam classes	8	20.0
Aminoglycosides + three beta-lactam classes	6	15.0
Aminoglycosides + fluoroquinolones + two beta-lactam classes	5	12.5
Three beta-lactam classes	3	7.5
Fluoroquinolones + two beta-lactam classes	2	5.0
XDR	28	12.5
Aminoglycosides + all beta-lactam classes	22	78.6
Aminoglycosides + fluoroquinolones + three beta-lactam classes	4	14.3
Fluoroquinolones + all beta-lactam classes	2	7.1
PDR	12	5.4

No piperacillin–tazobactam-resistant isolates.

MDR, multidrug resistant (R to three and four classes); PDR, pandrug resistant (R to all classes tested); XDR, extensively drug resistant (R to all but one class).

S. aureus accounted for 219 (40.4%) of all bacterial isolates from 113 patients with the incidence being stable between 38% and 43% throughout the study period (r = −0.53, p = 0.36). MDR-S. aureus (both MRSA and MDR-MSSA) incidence was 73.1% and MRSA accounted for 62.6% of all S. aureus isolated. MDR-MSSA was responsible for 10.5% S. aureus infections with the annual incidence increasing from 2.8% in 2010 to 12.5% in 2014 (r = 0.85, p = 0.071). MDR-MSSA isolates represented 28.0% of all MSSA isolates and were characterized by clindamycin, erythromycin, gentamicin, and trimethoprim/sulfamethoxazole resistance. Trimethoprim/sulfamethoxazole resistance was more common in MDR-MSSA isolates than both MSSA and MRSA isolates. There were no cases of vancomycin-intermediate or vancomycin-resistant S. aureus reported; however, overall incidence of MICs ≥1.5 μg/ml was 6.4%. Elevated vancomycin MICs became more common throughout the study period, starting at 2.8% of isolates in 2010, peaking at 13.6% in 2012, before settling at 8.5% in 2014 (r = 0.45, p = 0.45).

S. maltophilia was the third most commonly isolated organism with the incidence increasing from 6.7% in 2010 to 10.9% in 2014 (r = 0.84, p = 0.073). S. maltophilia was isolated from 32 patients (20.8%) during the study period. All Stenotrophomonas isolates were susceptible to minocycline, 91% was susceptible to trimethoprim/sulfamethoxazole, and only 62% was susceptible to levofloxacin.

Enterobacteriaceae were rarely isolated from CF cultures, accounting for only 34 isolates (6.3%) from 28 unique patients. Only two patients had multiple cultures for the same organism across the entire study period. One patient had a chronic polymicrobial infection with MDR-E. coli and P. aeruginosa from 2011 through 2014 and the other had Enterobacter cloacae with S. aureus in 2012 and 2014. Approximately, 41% of Enterobacteriaceae isolated were classified as multidrug resistant. Aside from cefazolin and piperacillin–tazobactam, beta-lactams were the most reliable class of agents across all Enterobacteriaceae isolates. MDR isolates were primarily resistant to fluoroquinolones, tetracycline, tobramycin, and trimethoprim–sulfamethoxazole.

Discussion

This study adds to the body of literature showing that P. aeruginosa and S. aureus comprise the majority of pathogens isolated from the respiratory tract of CF patients; therefore, empiric therapy of CF patients should include antipseudomonal and antistaphylococcal (specifically MRSA) agents. Although P. aeruginosa accounted for more respiratory isolates, S. aureus was present in 73.4% of patients compared to 68.2% for P. aeruginosa. S. maltophilia isolation increased dramatically over the study period and occurred in 20.8% of patients, concurring with Salsgiver et al.⁴ report.

In addition, we found a high rate of multidrug-resistant organisms present in CF sputum cultures compared to the published literature. In the 2013 Cystic Fibrosis Foundation (CFF) annual report,¹ the median percentage of patients with P. aeruginosa was 46.3% and 15.7% for MDR isolates. In our center, 68.2% of patients had at least one culture with P. aeruginosa and 31.8% of all patients had at least one culture with an MDR isolate. This seems indicative of the higher patient acuity at our center. It is important to recognize that we used a different definition for MDR than the CFF report; however, we feel that our definition is more liberal and likely resulted in more organisms being designated as MDR and is the more accepted definition.

This study is not without limitations. Primarily, this is a retrospective study of microbiological information with very little patient-specific data collected, and as such, recommendations for patient care may be limited. In addition, changing patient demographics and antibiotic exposure cannot be ruled out, which may bias the observed increase in MDR pathogens. Despite high rates of polymyxin susceptibility (100% of isolates tested) among P. aeruginosa isolates in this study, less than 25% of isolates were tested against these agents. Due to this finding, the polymyxins were exluded from the definitions of MDR. Emerging Gram-negative bacilli, such as Achromobacter spp. and Burkholderia cepacia complex, are commonly isolated from CF respiratory tract specimens.¹² In our population, data for these pathogens are sparse and antimicrobial resistance trends are infeasible with low isolate numbers. For this reason, data from emerging Gram-negative bacilli and Hemophilus influenzae are not presented.

MDR continues to increase in all types of infections; however, we have shown that specific CF pathogens display MDR at high frequencies. Due to the uncertainty regarding effective empiric therapy at our institution, empiric combination therapy should be utilized for antipseudomonal coverage in all CF patients.

Footnotes

Acknowledgments

The authors acknowledge Dr. Julie Ribas, MD, PhD, and the University of Kentucky HeathCare Clinical Microbiology laboratory for assistance in collection of data.

Disclosure Statement

No competing financial interests exist.

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