Molecular Characterization of Cotrimoxazole Resistance Genes and Their Associated Integrons in Clinical Isolates of Gram-Negative Bacteria from Tanzania

Abstract

Cotrimoxazole is widely used, particularly as a prophylactic drug in HIV patients. We assessed resistance mechanisms among cotrimoxazole resistant–Gram negative bacterial isolates (n = 123) obtained from blood (n = 69) and urine (n = 54) from Tanzanian patients. sul genes were detected in 98% (121/123) of the isolates. Coexistence of sul1 and sul2 was common (49/123). The dfr genes were found in 63% (77/123) of all isolates. sul1, dfrA15, and dfrA5 genes predominated among Klebsiella pneumoniae, while sul2 and dfrA1 genes were frequent in Escherichia coli isolates. Two isolates, both K. pneumoniae, carried sul3. Integrons were detected in 81.3% (100/123) of all isolates. Class 1 integrons were found in 95% (42/44), 53% (23/43), and 80.6% (25/31) of K. pneumoniae, E. coli, and other Enterobacteriaceae isolates, respectively. Class 2 integrons were found in 14% of E. coli, but not in K. pneumoniae. All sul1 genes in K. pneumoniae were carried in class 1 integrons. Gene cassette arrays dfrA5 and dfrA15-aadA1 were most frequently associated with class 1 integrons, while class 2 integrons contained only dfrA1-sat2-aadA1 gene cassettes. This is the first report of sul3 gene in K. pneumoniae from human sources. The finding that mechanisms differ between E. coli and K. pneumoniae may broaden our understanding of cotrimoxazole resistance.

Introduction

Cotrimoxazole is a combination of the antifolate compounds trimethoprim and sulfamethoxazole, which have a synergistic effect, and has been used as a broad spectrum antibiotic.¹ Cotrimoxazole has been used for several decades as an efficient antibiotic of choice for treatment of enteric bacterial infections, urinary tract infections, respiratory tract infections, and skin and soft tissue infections.² The drug is extensively used in resource poor settings because it is relatively cheap, available over-the-counter, and well tolerated.³ Furthermore, during the past two decades, cotrimoxazole has been widely used as a prophylactic drug in HIV/AIDS patients as part of a package of HIV care and treatment. The injudicious use of cotrimoxazole in developing countries has been a major factor in the emergence of bacteria with decreased susceptibility to this antibiotic.^3,4 Recent studies from the African continent have reported high rates of cotrimoxazole resistance in Gram-negative pathogenic bacteria in the range of 50–96%.^5–7 Likewise, in Tanzania, studies have showed that 67–97% of Gram-negative bacteria pathogens from bloodstream infections, urinary tract infections, and surgical site infections were resistant to cotrimoxazole.^8,9

The constituents of cotrimoxazole, sulfamethoxazole and trimethoprim, inhibit the enzymes dihydropteroate synthase (DHPS) and dihydrofolate reductase, respectively. Resistance in enteric Gram-negative bacteria generally arises from plasmid-mediated acquisition of variants of these enzymes, which are not inhibited by the drugs.² Three plasmid-mediated DHPS genes encoding sulfonamide resistance, sul1, sul2, and sul3, have been identified in Gram-negative bacteria,^10,11 and more than 30 dfr gene encoding resistant variants to trimethoprim have been described.¹⁰

Integrons are implicated in the development and spread of antibiotic resistance among Gram-negative bacteria.¹² sul1 and dfr genes in Enterobacteriaceae are often associated with class 1 integrons residing on plasmids, transposons, and/or bacterial chromosomes.^10,12 sul2 is usually located on small nonconjugative plasmids, but has recently been found located on large conjugative plasmids.^12,13 The third type of sulfonamide resistance genes, sul3, which has mainly been found in animals,^14,15 was first described in Escherichia coli from a human clinical sample from Sweden in 2003¹⁶ and is now occasionally found in human E. coli isolates.^17,18

While resistance to cotrimoxazole is rampant in resource-limited settings, only one study from Central African Republic (CAR) and two from Tunisia have reported on the mechanisms of resistance in samples from the African continent.^19–21 No data are available from Tanzania although there is massive use of cotrimoxazole both as general antibiotic treatment and as prophylactic therapy in people with HIV infection. Therefore, we aimed to investigate the molecular basis of cotrimoxazole resistance among Gram-negative bacteria isolated at Muhimbili National Hospital (MNH), Dar es Salaam, Tanzania.

Materials and Methods

Study population, design, and setting

Two study populations were involved in the current study. The first was from a cross-sectional study, which was performed between June 2004 and January 2005 at MNH. In this study population, patients fulfilling the criteria for urinary tract infections were consecutively enrolled as community/outpatients and inpatients. Community/outpatients were nonhospitalized patients from the community and inpatients were hospitalized patients at MNH. The second study was a prospective analysis of the bacteria isolated from pediatric bloodstream infections,²² conducted at the same hospital from August 2001 to August 2002. MNH, the largest hospital in Tanzania, is the national referral hospital and serves a tertiary healthcare facility for a population of over 4 million residents living in Dar es Salaam.

Bacterial isolates

From the first study population, 54 nonduplicate cotrimoxazole resistant–Gram negative bacterial isolates were collected from positive urine cultures of patients with significant bacteriuria (>10⁵ cfu/ml). From the second study population, we included 69 nonduplicate cotrimoxazole resistant–Gram negative bacterial isolates from pediatric bloodstream infections. All isolates were identified to the species level by standard methods,²³ API20E (bioMérieux SA, Marcy l'Etoile, France) and the VITEK 2 system (bioMérieux, Inc., Durham, NC).

Antimicrobial susceptibility testing

Antibiotic susceptibility was determined by the disk diffusion method according to National Committee for Clinical Laboratory Standards (NCCLS; currently Clinical and Laboratory Standards Institute [CLSI]) guidelines.²⁴ Minimum inhibitory concentration (MIC) test strips (Liofilchem, Roseto Degli Abruzzi, Italy) were used for determination of cotrimoxazole MIC according to the manufacturer's instructions and interpreted in accordance to CLSI guidelines.²⁵ E. coli ATCC 25922 was used as control strain. For antimicrobial susceptibility results, see Supplementary Table S1 (Supplementary materials are available online at www.liebertpub.com/mdr).

Isolation of genomic DNA

Genomic DNA was isolated by a rapid boiling procedure and stored at −20°C.

Detection of sulfonamide resistance genes (sul)

Conventional multiplex PCR was used for detection of sul1, sul2, and sul3 genes with primers and PCR conditions described previously.^26–28 PCR was performed using QIAGEN Multiplex PCR Kit (Qiagen, Mississauga, ON, Canada) and GeneAmp 9700 Thermocycler (Applied Biosystems, Foster City, CA). The reaction mixture contained 1× Qiagen multiplex PCR master mix, 5 μl 1× Q-solution, 0.2 μM each of sul1 and sul3 primers, 0.3 μM sul2 primers, 2.5 μl of DNA template, and water to a total volume of 25 μl. The amplicons were analyzed by agarose gel electrophoresis. E. coli isolates carrying sul1, sul2, and sul3 were used as positive controls, and RNAse-free water was used as negative control.

Detection of trimethoprim resistance genes (dfr)

Trimethoprim resistance genes dfrA1, dfrA5, and dfrA12 were detected by real-time PCR using a LightCycler 480 Instrument II (Roche Diagnostics, Basel, Switzerland) and using primers and PCR conditions as described by Grape et al.²⁹ E. coli isolates carrying dfrA1, dfrA5, and dfrA12 were used as positive controls.

Integrons and their variable region characterization

Presence of class 1 integrons (intI1) was investigated by PCR using primers and conditions as described by Frank et al.¹⁹ Characterization of class 2 integrons (intI2) and variable regions (gene cassettes) of class 1 and 2 integrons were performed by conventional PCR using primers and conditions as described by Zeighami et al.³⁰ PCR was performed on a GeneAmp 9700 Thermocycler (Applied Biosystems). A final reaction volume of 25 μl consisted of the following: 1× HotStarTaq Plus Master Mix (QIAGEN, Hilden, Germany), 10 pmol of each primer, 8.5 μl RNAse-free water, and 2.5 μl of DNA template. The amplified products were analyzed by gel electrophoresis.

Sequencing of sul3 and integron variable regions (gene cassettes)

The PCR products of sul3 gene and the variable regions of integrons were purified and both strands sequenced, using the same primers as for PCRs. Sequencing was performed using BigDye Terminator v1.1 Cycle Sequencing Kit (Applied Biosystems) and an ABI PRISM 3730 DNA Analyzer (Applied Biosystems). Sequences were identified using the Basic Local Alignment Search Tool (BLAST) program available at the website of the National Center for Biotechnology Information (NCBI; http://blast.ncbi.nlm.nih.gov/Blast.cgi).

Data analysis

Statistical analysis was performed using SPSS software version 20.0 (IBM SPSS statistics 20.0; SPSS, Inc., Chicago, IL). Chi-square test was used to determine the association of the data; p < 0.05 was considered significant.

Ethical approval

In Tanzania, ethical approvals to conduct both studies were obtained from senate research and publication committee of Muhimbili University of Health and Allied Sciences, Dar es Salaam, Tanzania. In Norway, the ethical approval to conduct urinary tract infections was sought from Regional Committee for Ethics in Health Research of Western Norway. For bloodstream infection study, ethical approval was sought from REK Vest, Norway.

Permission was obtained from MNH authorities. Written informed consent was obtained from patients in urinary tract infection study and from parents/caretakers of children in bloodstream infection study.

Results

Bacterial isolates

Of 123 Gram-negative bacteria investigated, Klebsiella pneumoniae (n = 44) and E. coli (n = 43) were the most prevalent isolates. Other Enterobacteriaceae (Enterobacter cloacae, Proteus mirabilis, Morganella morganii, Citrobacter freundii, and Providencia rettgeri) accounted for 31 isolates and 5 were Gram-negative rods other than Enterobacteriaceae (Acinetobacter baumannii [1] and Pseudomonas aeruginosa [4]).

Prevalence of sulfonamide resistance genes (sul genes)

The distribution of sul genes is shown in Table 1. We detected sul genes in 121 out of 123 (98.4%) cotrimoxazole-resistant isolates, whereas sul genes were not detected in one K. pneumoniae isolate and one P. aeruginosa isolate.

Table 1.

Distribution of Integrons and Cotrimoxazole Resistance Genes Among Cotrimoxazole-Resistant Isolates

	Sul					dfr									intI
	sul1	sul2	sul1 + 2	Sul1 + 3	No sul	dfrA1	dfrA5	dfrA12	dfrA7	dfrA17	dfrA1 + 5	dfrA1 + 12	dfrA15	No dfr	intI1	intI2	intI1 + 2	No intI
Klebsiella pneumoniae (44)	21	9	11	2	1	2	12	2	1	0	1	1	16	9	42	0	0	2
Escherichia coli (43)	0	24	19	0	0	9	4	1	8	4	1	0	0	16	23	5	1	14
Enterobacter cloacae (13)	4	1	8	0	0	2	0	2	0	0	0	0	0	9	11	0	0	2
Proteus mirabilis (6)	2	0	4	0		3	0	0	1	0	0	0	1	1	5	0	0	1
Morganella morganii (6)	4	0	2	0	0	1	0	0	0	1	0	0	1	3	5	0	0	1
Citrobacter freundii (4)	2	1	1	0	0	0	1	1	0	0	0	0	0	2	3	0	0	1
P. aeruginosa (4)	2	0	1	0	1	0	0	0	0	0	0	0	0	4	2	0	0	2
Providencia rettgeri (2)	0	0	2	0	0	1	0	0	0	0	0	0	0	1	1	0	1	0
Acinetobacter baumannii (1)	0	0	1	0	0	0	0	0	0	0	0	0	0	1	1	0	0	0
Total	35	35	49	2	2	18	17	6	10	5	2	1	18	46	93	5	2	23

The coexistence of sul1 and sul2 genes was found in 40.5% (49/121) of the isolates. We found equal prevalence, 28.9% (35/121), of isolates carrying either sul1 only or sul2 only. Two bacterial isolates carried sul3. These were both K. pneumoniae, in which sul3 coexisted with sul1. As seen in Table 1, sul1 gene predominated among K. pneumoniae isolates with 77% (34/44) (p < 0.05) compared to E. coli with 44% (19/43). In contrast, sul2 gene predominated among E. coli isolates with 100% (43/43) (p < 0.05) compared to K. pneumoniae with 25% (11/44).

Prevalence of trimethoprim resistance genes (dfr genes)

Table 1 also shows the distribution of dfr genes. The dfr genes were detected in 77 out of 123 bacterial isolates (62.6%). The most prevalent dfr gene was dfrA1, followed by dfrA5, dfrA15, and dfrA7.

The dfr genes were detected in 78% (35/44) of K. pneumoniae, 63% (27/45) of E. coli, and 47% (15/31) among other Enterobacteriaceae. The dfrA15, followed by dfrA5, predominated in K. pneumoniae, while dfrA1 and dfrA7 predominated among E. coli isolates. The dfr genes were not detected in any of the A. baumannii or P. aeruginosa isolates. We found coexistence of dfrA1 and dfrA5 in one K. pneumoniae isolate and one E. coli isolate. The coexistence of dfrA1 and dfrA12 was also found in one K. pneumoniae isolate.

Integron carriage and variable region characterization

Integrons were detected in 81.3% (100/123) of the cotrimoxazole-resistant isolates. The prevalence of class 1 and 2 integrons was 75.6% (93/123) and 4.1% (5/123), respectively, whereas two isolates had both class 1 and 2 integrons of 1.6% (2/123).

Class 1 integrons were predominantly detected in K. pneumoniae with 95% (42/44) (p < 0.05) than 53% (23/43) in E. coli. All five class 2 integrons detected were found in E. coli isolates. One E. coli isolate and one P. rettgeri isolate carried both class 1 and 2 integrons.

The characterization of integron variable regions revealed the presence of 81 gene cassettes arranged in 22 different gene cassette arrays as shown in Table 2. The most prominent gene cassette arrays were dfrA5 (n = 16), dfrA15-aadA1 (n = 16), and dfrA7 (n = 10).

Table 2.

Distribution of Integrons and Gene Cassettes Among Cotrimoxazole-Resistant Isolates

Isolates (n)	intI (n)	Gene cassette	n
K. pneumoniae (44)	intI1 (42)	dfr15-aadA1	15
		dfrA5	12
		dfrA1-dfrA5	1
		dfrA7	1
		dfrA12-orfF-aadA2	2
		dfrA1 (dfrA12-orfF-aadA2)	1
		dfrA1	1
		dfrA15	1
		dfrA1-aadA1	1
	No intI (2)	No gene cassette	0
E. coli (43)	intI1 (23)	dfrA7	7
		dfrA5	1
		dfrA1,dfrA5	1
		dfr17	1
		dfrA12-orfF-aadA2	1
		dfrA1-aadA1	1
		dfrA17-aadA5	2
	intI2 (5)	dfrA1-sat2-aadA1	5
	intI1 + 2 (1)	dfrA1-sat2-aadA1	1
	No intI (14)	dfrA5	3
		dfrA7	1
		dfrA17-aadA5	1
		dfrA1	2
Enterobacteriaceae	intI1 (25)	dfrA7	1
		dfrA12-orfF-aadA2	2
		dfrA17-aadA5	1
		dfrA1	1
		dfrA15	2
		aadA1	1
		aadA2	1
		dfrA5-aadA2	1
		dfrA1-orfC	1
		aacA4	1
		dfrA1-aacA4	1
		aadB-catB3	1
	intI1+intI2 (1)	dfrA1	1
	No intI (5)	dfrA12-orfF-aadA2	1
		dfrA1	2
Other GNR	intI1 (3)	aadB	1
	No intI1 (2)	aacA4	1
Total			81

GNR, Gram-negative rods; intI, Integron.

The most prevalent gene cassettes associated with class 1 integron in K. pneumoniae were dfrA15-aadA1 (n = 16) and dfrA5 (n = 12), while in E. coli it was dfrA7 (n = 7). All E. coli carrying class 2 integrons contained only dfrA1-sat2-aadA1 gene cassettes (encoding resistance to trimethoprim, streptothricin, and aminoglycosides). Eleven of the gene cassettes were not associated with any integron structures, 7 were E. coli (3 carried dfrA5, 2 dfrA1, 1 dfrA7, and 1 dfrA17-aadA5). Two were E. cloacae (one dfrA1, one dfrA12-orfF-aadA2), one P. mirabilis (dfrA1), and one P. aeruginosa (aacA4).

Aminoglycoside resistance gene cassettes (aadA1 and aadA2) were detected in 19 K. pneumoniae isolates and in 11 E. coli isolates.

Discussion

Cotrimoxazole has been widely used in Africa as a first line drug and for prophylaxis for HIV-infected people. This is thought to be the driving force toward the emergence and spread of cotrimoxazole resistance among Gram-negative bacteria. However, only few studies on genetic mechanisms underlying cotrimoxazole resistance have been published from the African continent.^19–21 This is the first study from Tanzania to assess genetic mechanisms of cotrimoxazole resistance in clinical bacterial isolates. We found differences in the occurrence of sul genes, dfr genes, and integron types between K. pneumoniae, E. coli, other Enterobacteriaceae, and non-Enterobacteriaceae.

Little is known about sul genes in K. pneumoniae.^31,32 In this study, we found that predominant sul genes differ between K. pneumoniae and E. coli. To date, in humans, sul3 genes have only been reported in E. coli from urine and fecal commensals in Europe, America, and Asia.^16,17,32,33 Previously, sul3 genes were reported in E. coli isolates from pigs, cattle, and poultry.^14,15 To our knowledge, this is the first report on detection of sul3 gene from human sources in Africa. It is also the second report of the presence of sul3 gene in Africa. A recent study in Tunisia reported for the first time the presence of sul3 gene in E. coli isolates from poultry meat.²¹ This may indicate extensive spread of this resistance determinant. The two K. pneumoniae isolates with sul3, also carrying the sul1 gene and class 1 integrons, were from hospital- and community-acquired urinary tract infections. Furthermore, one contained dfrA12-orfF-aadA2 gene cassette, but the other isolate had no dfr gene. The diverse origin of the isolates and variation observed might suggest the presence of two diverse clones.

The sul1 gene was more common in K. pneumoniae and other Enterobacteriaceae than in E. coli. The sul2 gene was most frequent in E. coli. The finding of higher prevalence of sul2 than sul1 in E. coli isolates from humans has also been observed previously in the CAR,¹⁹ as well as from Europe and elsewhere.^17,18,33 The association of sul1 gene as part of the 3′-conserved-segment (3′-CS) of class 1 integrons has been well documented before.¹² We also found all K. pneumoniae harboring sul1 gene to be associated with class 1 integrons, in contrast to what we observed in E. coli. Seven E. coli isolates, which carried sul1 gene, did not have class 1 integrons, possibly indicating the presence of sul1 alleles positioned on a different genetic element. In contrast, 20 isolates positive for class 1 integron were negative for the sul1 gene, while all were positive for the sul2 gene. Lack of the sul1 gene on class 1 integron structure has been reported before in E. coli isolates from humans and animals.^34,35 The sul2 gene has no known association with any integron structures, implying other mechanisms. However, we observed that all E. coli positive for class 2 integrons were also positive for the presence of sul2 gene only. Our study did not investigate further regarding the localization of sul2 genes on plasmids. The finding of low prevalence of class 1 integron in E. coli compared to K. pneumoniae may partly explain the observed differences of predominant sul genes in different bacteria.

This study also found variation in carriage of trimethoprim resistance genes in different types of bacteria. The dfrA15 and dfrA5 genes were frequently detected in K. pneumoniae isolates while dfrA1 and dfrA7 genes were common in E. coli. Our finding of the predominance of dfrA1 and dfrA7 in E. coli is in contrast to earlier studies performed in Africa and a recent study from South Korea, which found that dfrA17, dfrA7, and dfrA5 were the most common genes.^19,20,36 A recent study from Iran found dfrA1 and dfrA17 to be the most common genes in uropathogenic E. coli.³⁰ The reason for variation of the predominant dfr gene in E. coli is not well known. Horizontal transfer and clonal spread of the isolates carrying dfr genes in a certain region could be a possibility. Furthermore, we observed that the predominant dfr genes in K. pneumoniae were different from those in E. coli. The dfrA15, which was the most common in K. pneumoniae, was not found in E. coli, and dfrA17, which was third most common in E. coli, was not detected in K. pneumoniae. Our results concur with a recent study from Sweden, which found that different mechanisms of dfr gene transfer exist between E. coli and K. pneumoniae.³¹ Furthermore, we found coexistence of dfr genes in three isolates. It is rare for one isolate to carry more than one dfr gene, but this has been described previously in Europe and Korea.³⁷

We found a high frequency of carriage of class 1 integron among cotrimoxazole-resistant bacteria, which is in line with previous studies from Africa and elsewhere.^18–20 The prevalence of class 1 integrons in E. coli in our study is similar to the findings in a study of urinary tract isolates from Korea.³⁸ Linkage of class 1 integrons and antimicrobial resistance has been well established. Our finding of low prevalence of class 1 integrons in E. coli in comparison to K. pneumoniae and other Enterobacteriaceae is in contrast to a recent study performed in Sweden.³¹ In Sweden, class 1 integrons were more prevalent in E. coli than in K. pneumoniae. In this study, some bacteria with dfr and sul genes were lacking integrons. These resistance genes could be from genetic elements other than integrons, as described by other studies.^11,20

We found dfr and aadA to be the most frequent gene cassettes associated with class 1 integrons, in line with previous studies from Africa, Europe, and Asia.^20,30,34 We also found predominance of single gene cassettes compared to multigene cassettes. In contrast to previous studies,^32,35 we found that the dfrA7 was the predominant class 1 integron gene cassette array in E. coli isolates. Meanwhile, in K. pneumoniae, dfrA15-aadA1 was the most prevalent gene cassette array associated with class 1 integron, which is inconsistent to previous studies.^32,39 In accordance with previous studies,^34,40 we found that class 2 integron carried only dfrA1-sat2-aadA1 gene cassettes. The finding of low prevalence of class 2 integron in our study is in line with previous studies performed in Europe and Asia.^34,37

Furthermore, we found 11 gene cassettes not associated with any integron structures. In the findings which were similar to the study, we used the primers and PCR conditions.²⁰ This phenomena could be explained by the failure of the PCR to recognize and/or deletion of intI genes.

One of the limitations of the study is that the isolates analyzed were isolated 11 years ago. Thus, some caution is necessary in interpretation of dissemination of sul and dfr genes, as well as distribution of integrons. Results may not imply the current gene dissemination in the study setting. Another limitation is that epidemiological typing was not investigated, which could have shed more light on genetic relatedness of the isolates.

In conclusion, we found a high prevalence of both sul and dfr genes among Gram-negative bacterial isolates from Tanzania. We report for the first time the presence of sul3 gene in K. pneumoniae from human sources from Africa. Integrons were widely disseminated in our isolates, but not similarly distributed in different bacterial species, indicating that horizontal transfer between species is a rare occurrence. In our study, the mechanisms of resistance to cotrimoxazole were different between E. coli and K. pneumoniae. Therefore, relying on one bacterial species, that is, E. coli, to understand underlying mechanisms of resistance to cotrimoxazole may be insufficient.

Footnotes

Acknowledgments

The authors thank the technical staff of the Department of Microbiology at Muhimbili National Hospital, Dar es Salaam, Tanzania, for their valuable support during specimen processing. The authors also acknowledge the members of the Department of Microbiology and Immunology, Haukeland University Hospital, Bergen, Norway, and the Department of Clinical Science, University of Bergen, Bergen, Norway, for their technical support during the molecular study. Finally, the authors acknowledge Ørjan Samuelsen, University of Tromsø, Tromsø, Norway, for providing control strains.

Disclosure Statement

No competing financial interests exist.

References

Rubin

R.H.

, and Swartz

M.N.

1980. Trimethoprim-sulfamethoxazole. N. Engl. J. Med., 303:426–432.

Huovinen

2001. Resistance to trimethoprim-sulfamethoxazole. Clin. Infect. Dis., 32:1608–1614.

Morgan

D.J.

, Okeke

I.N.

, Laxminarayan

, Perencevich

E.N.

, and Weisenberg

2011. Non-prescription antimicrobial use worldwide: a systematic review. Lancet Infect. Dis., 11:692–701.

Okeke

I.N.

, Lamikanra

, and Edelman

1999. Socioeconomic and behavioral factors leading to acquired bacterial resistance to antibiotics in developing countries. Emerg. Infect. Dis., 5:18–27.

Langendorf

, Le Hello

, Moumouni

, Gouali

, Mamaty

A.A.

, Grais

R.F.

, Weill

F.X.

, and Page

A.L.

2015. Enteric bacterial pathogens in children with diarrhea in Niger: diversity and antimicrobial resistance. PLoS One, 10:e0120275.

Ntirenganya

, Manzi

, Muvunyi

C.M.

, and Ogbuagu

2015. High prevalence of antimicrobial resistance among common bacterial isolates in a tertiary healthcare facility in Rwanda. Am. J. Trop. Med. Hyg., 92:865–870.

Isendahl

, Manjuba

, Rodrigues

, Xu

, Henriques-Normark

, Giske

C.G.

, and Naucler

2014. Prevalence of community-acquired bacteraemia in Guinea-Bissau: an observational study. BMC Infect. Dis., 14:3859.

Kayange

, Kamugisha

, Mwizamholya

D.L.

, Jeremiah

, and Mshana

S.E.

2010. Predictors of positive blood culture and deaths among neonates with suspected neonatal sepsis in a tertiary hospital, Mwanza-Tanzania. BMC Pediatr., 10:39.

Manyahi

, Matee

M.I.

, Majigo

, Moyo

, Mshana

S.E.

, and Lyamuya

E.F.

2014. Predominance of multi-drug resistant bacterial pathogens causing surgical site infections in Muhimbili National Hospital, Tanzania. BMC Res. Notes, 7:500.

10.

Huovinen

, Sundstrom

, Swedberg

, and Skold

1995. Trimethoprim and sulfonamide resistance. Antimicrob. Agents Chemother., 39:279–289.

11.

Gundogdu

, Long

Y.B.

, Vollmerhausen

T.L.

, and Katouli

2011. Antimicrobial resistance and distribution of sul genes and integron-associated intI genes among uropathogenic Escherichia coli in Queensland, Australia. J. Med. Microbiol., 60(Pt 11):1633–1642.

12.

P.L.

, Wong

R.C.

, Chow

K.H.

, and Que

T.L.

2009. Distribution of integron-associated trimethoprim-sulfamethoxazole resistance determinants among Escherichia coli from humans and food-producing animals. Lett. Appl. Microbiol., 49:627–634.

13.

Bean

D.C.

, Livermore

D.M.

, and Hall

L.M.

2009. Plasmids imparting sulfonamide resistance in Escherichia coli: implications for persistence. Antimicrob. Agents Chemother., 53:1088–1093.

14.

Perreten

, and Boerlin

2003. A new sulfonamide resistance gene (sul3) in Escherichia coli is widespread in the pig population of Switzerland. Antimicrob. Agents Chemother., 47:1169–1172.

15.

Guerra

, Junker

, Schroeter

, Malorny

, Lehmann

, and Helmuth

2003. Phenotypic and genotypic characterization of antimicrobial resistance in German Escherichia coli isolates from cattle, swine and poultry. J. Antimicrob. Chemother., 52:489–492.

16.

Grape

, Sundstrom

, and Kronvall

2003. Sulphonamide resistance gene sul3 found in Escherichia coli isolates from human sources. J. Antimicrob. Chemother., 52:1022–1024.

17.

Infante

, Grape

, Larsson

, Kristiansson

, Pallecchi

, Rossolini

G.M.

, and Kronvall

2005. Acquired sulphonamide resistance genes in faecal Escherichia coli from healthy children in Bolivia and Peru. Int. J. Antimicrob. Agents, 25:308–312.

18.

Blahna

M.T.

, Zalewski

C.A.

, Reuer

, Kahlmeter

, Foxman

, and Marrs

C.F.

2006. The role of horizontal gene transfer in the spread of trimethoprim-sulfamethoxazole resistance among uropathogenic Escherichia coli in Europe and Canada. J. Antimicrob. Chemother., 57:666–672.

19.

Frank

, Gautier

, Talarmin

, Bercion

, and Arlet

2007. Characterization of sulphonamide resistance genes and class 1 integron gene cassettes in Enterobacteriaceae, Central African Republic (CAR). J. Antimicrob. Chemother., 59:742–745.

20.

Dahmen

, Mansour

, Boujaafar

, Arlet

, and Bouallegue

2010. Distribution of cotrimoxazole resistance genes associated with class 1 integrons in clinical isolates of Enterobacteriaceae in a university hospital in Tunisia. Microb. Drug Resist., 16:43–47.

21.

Soufi

, Abbassi

M.S.

, Saenz

, Vinue

, Somalo

, Zarazaga

, Abbas

, Dbaya

, Khanfir

, Ben Hassen

, Hammami

, and Torres

2009. Prevalence and diversity of integrons and associated resistance genes in Escherichia coli isolates from poultry meat in Tunisia. Foodborne Pathog. Dis., 6:1067–1073.

22.

Blomberg

, Manji

K.P.

, Urassa

W.K.

, Tamim

B.S.

, Mwakagile

D.S.

, Jureen

, Msangi

, Tellevik

M.G.

, Holberg-Petersen

, Harthug

, Maselle

S.Y.

, and Langeland

2007. Antimicrobial resistance predicts death in Tanzanian children with bloodstream infections: a prospective cohort study. BMC Infect. Dis., 7:43.

23.

Collee

J.G.

, Marmion

B.P.

, Irvine

, Fraser

A.G.

, and Simmons

1996. Mackie & McCartney Practical Medical Microbiology. 14th ed. Churchill Livingstone, New York.

24.

National Committee for Clinical Laboratory Standards (NCCLS). 2002. Performance Standards for Antimicrobial Susceptibility Testing: Twelfth Informational Supplement M100-S12. NCCLS, Wayne, PA.

25.

Clinical and Laboratory Standards Institute (CLSI). 2010. Performance Standards for Antimicrobial Susceptibility Testing: Twentieth Informational Supplement M100-S20. CLSI, Wayne, PA.

26.

Kerrn

M.B.

, Klemmensen

, Frimodt-Moller

, and Espersen

2002. Susceptibility of Danish Escherichia coli strains isolated from urinary tract infections and bacteraemia, and distribution of sul genes conferring sulphonamide resistance. J. Antimicrob. Chemother., 50:513–516.

27.

Lanz

, Kuhnert

, and Boerlin

2003. Antimicrobial resistance and resistance gene determinants in clinical Escherichia coli from different animal species in Switzerland. Vet. Microbiol., 91:73–84.

28.

Kozak

G.K.

, Boerlin

, Janecko

, Reid-Smith

R.J.

, and Jardine

2009. Antimicrobial resistance in Escherichia coli isolates from swine and wild small mammals in the proximity of swine farms and in natural environments in Ontario, Canada. Appl. Environ. Microbiol., 75:559–566.

29.

Grape

, Motakefi

, Pavuluri

, and Kahlmeter

2007. Standard and real-time multiplex PCR methods for detection of trimethoprim resistance dfr genes in large collections of bacteria. Clin. Microbiol. Infect., 13:1112–1118.

30.

Zeighami

, Haghi

, Masumian

, Hemmati

, Samei

, and Naderi

2015. Distribution of integrons and gene cassettes among uropathogenic and diarrheagenic Escherichia coli isolates in Iran. Microb. Drug Resist., 21:435–440.

31.

Brolund

, Sundqvist

, Kahlmeter

, and Grape

2010. Molecular characterisation of trimethoprim resistance in Escherichia coli and Klebsiella pneumoniae during a two year intervention on trimethoprim use. PLoS One, 5:e9233.

32.

Shin

H.W.

, Lim

, Kim

, Kwon

G.C.

, and Koo

S.H.

2015. Characterization of trimethoprim-sulfamethoxazole resistance genes and their relatedness to class 1 integron and insertion sequence common region in gram-negative bacilli. J. Microbiol. Biotechnol., 25:137–142.

33.

, Dalsgaard

, Hammerum

A.M.

, Porsbo

L.J.

, and Jensen

L.B.

2010. Prevalence and characterization of plasmids carrying sulfonamide resistance genes among Escherichia coli from pigs, pig carcasses and human. Acta Vet. Scand., 52:47.

34.

Vinue

, Saenz

, Somalo

, Escudero

, Moreno

M.A.

, Ruiz-Larrea

, and Torres

2008. Prevalence and diversity of integrons and associated resistance genes in faecal Escherichia coli isolates of healthy humans in Spain. J. Antimicrob. Chemother., 62:934–937.

35.

Sunde

2005. Prevalence and characterization of class 1 and class 2 integrons in Escherichia coli isolated from meat and meat products of Norwegian origin. J. Antimicrob. Chemother., 56:1019–1024.

36.

Gassama

, Aidara-Kane

, Chainier

, Denis

, and Ploy

M.C.

2004. Integron-associated antibiotic resistance in enteroaggregative and enteroinvasive Escherichia coli. Microb. Drug Resist., 10:27–30.

37.

Lee

J.C.

, Oh

J.Y.

, Cho

J.W.

, Park

J.C.

, Kim

J.M.

, Seol

S.Y.

, and Cho

D.T.

2001. The prevalence of trimethoprim-resistance-conferring dihydrofolate reductase genes in urinary isolates of Escherichia coli in Korea. J. Antimicrob. Chemother., 47:599–604.

38.

H.S.

, Lee

J.C.

, Kang

H.Y.

, Jeong

Y.S.

, Lee

E.Y.

, Choi

C.H.

, Tae

S.H.

, Lee

Y.C.

, Seol

S.Y.

, and Cho

D.T.

2004. Prevalence of dfr genes associated with integrons and dissemination of dfrA17 among urinary isolates of Escherichia coli in Korea. J. Antimicrob. Chemother., 53:445–450.

39.

, Hu

, Wang

, Yi

, Woo

P.C.

, Jing

, Zhu

, and Liu

C.H.

2013. Structural diversity of class 1 integrons and their associated gene cassettes in Klebsiella pneumoniae isolates from a hospital in China. PLoS One, 8:e75805.

40.

Xia

, Ren

, Guo

, and Xu

2013. Molecular diversity of class 2 integrons in antibiotic-resistant gram-negative bacteria found in wastewater environments in China. Ecotoxicology, 22:402–414.

Supplementary Material

Please find the following supplemental material available below.

For Open Access articles published under a Creative Commons License, all supplemental material carries the same license as the article it is associated with.

For non-Open Access articles published, all supplemental material carries a non-exclusive license, and permission requests for re-use of supplemental material or any part of supplemental material shall be sent directly to the copyright owner as specified in the copyright notice associated with the article.

0.02 MB

0.00 MB