Presence of OXA-23-Producing Isolates of Acinetobacter baumannii in Wastewater from Hospitals in Southern Brazil

Abstract

The aim of the study was to evaluate the dissemination of multiresistant isolates of Acinetobacter baumannii carrying resistance genes, by samples of wastewater from hospitals in Porto Alegre, Rio Grande do Sul, Brazil. We obtained 303 bacterial isolates from the wastewater of three hospitals in Porto Alegre, Rio Grande do Sul. For each isolate, we determined the profile of susceptibility to antimicrobials and the presence of the genes bla_OXA-23, bla_OXA-24, bla_OXA-51, bla_OXA-58, bla_SPM-1, bla_IMP, and bla_VIM. The bla_OXA-51 gene was found in 56% of the isolates, indicating the presence of A. baumannii in this environment. Of these, three multiresistant isolates were positive for the bla_OXA-23 gene, in wastewater from two of the hospitals. The results obtained in this study indicate that isolates of A. baumannii which are multiresistant and carry resistance genes such as bla_OXA-51 and bla_OXA-23 are being released into the environment in the wastewater from the hospitals analyzed. Multiresistant Acinetobacter junii, the newly emerging pathogen, were also found among the multiresistant isolates. Hospital wastewater may be crucial to the development and dispersal of multiresistant bacteria, making waterbodies reservoirs of bacterial resistance.

Introduction

Pathogenic bacteria of humans and animals are being constantly released into waterbodies via wastewater.³ The hospital environment is considered highly selective for antimicrobial-resistant strains, which can make hospital wastewater a vector for dissemination of multiresistant bacteria.¹⁷ Many of these microorganisms carry resistance genes, which are occasionally inserted in movable regions of the bacterial DNA. The acquisition of these determinants of antimicrobial resistance by horizontal transfer has a large role in the development and dispersal of resistance among pathogenic bacteria.¹ Water is not only a route for the dissemination of resistant microorganisms among human and animal populations but also a route for the dissemination of resistance genes in natural bacterial communities. In these communities, nonpathogenic bacteria may serve as a reservoir for resistance genes. In addition to the introduction of antimicrobials via hospital wastewater into waterbodies, detergents, disinfectants, and industrial residues such as heavy metals contribute to the evolution and dispersal of these resistant microorganisms in the aquatic environment.³

The most prominent examples of selection of microorganisms that are intrinsically resistant in the environment are the Gram-negative opportunist pathogens Acinetobacter spp., Pseudomonas aeruginosa, and Burkholderia cepacia, which are characteristic of soil.¹ The genus Acinetobacter is widely distributed and is capable of surviving in adverse environments, and it also shows great ability to rapidly acquire resistance to antibiotics.^4,22 In recent years, the genus Acinetobacter has been associated with a large number of outbreaks in hospitals worldwide,^14,23,29,35 including Brazil.^7,8,11,25,26

In many cases, carbapenems have been the sole drug of choice for the treatment of infections caused by multiresistant Acinetobacter spp. Among the mechanisms of resistance to carbapenems is the production of carbapenemases of classes B and D, according to the classification of Ambler.^21,28 Class B enzymes (metallo-β-lactamases [MBL]) reside in movable regions of bacterial DNA, as do some class D enzymes, which can make these genes transferable to other bacteria. The bla_OXA-23 gene, a member of Class D, is located either in a plasmid or is chromosomal encoded, as in Acinetobacter radioresistens sp. This enzyme has been associated with various outbreaks of infection in different parts of the world¹⁸ and has shown clonal dissemination in studies in Brazilian hospitals.^7,8

Studies on antimicrobial resistance normally focus on bacterial isolates obtained in clinical samples. However, genetic determinants of resistance are present in the microbial communities of natural environments and, thus, may be selected and disseminated by means of untreated wastewater and directly released into waterbodies. In view of the existence of transferable determinants of resistance and the potential of the genus Acinetobacter to exchange genetic material, the objective of this study was to evaluate the dissemination of isolates of multiresistant strains of Acinetobacter baumannii in samples from hospital wastewater in the city of Porto Alegre, Rio Grande do Sul, and to determine the presence of bla_OXA-23, bla_OXA-24, bla_OXA-51, bla_OXA-58, bla_SPM-1, bla_IMP, and bla_VIM genes.

Materials and Methods

Samples

Samples were collected twice from the wastewater of three large hospitals in Porto Alegre, RS, Brazil. At Hospital de Clínicas de Porto Alegre, samples were collected in March and August 2007; at Hospital São Lucas, in July 2006 and August 2007; and at Hospital Conceição, in November 2006 and July 2007. Untreated wastewater sampling was performed at the end of the hospital-building networks, and the sampling was randomly done. Hospital São Lucas has 539 beds and a circulation of 18,000 persons/day; Hospital de Clínicas de Porto Alegre has 714 beds and 12,000 persons/day; and Hospital Conceição has 840 beds and 21,000 persons/day.

Samples of 1 L of wastewater were collected, and aliquots of 100 ml were filtered on mixed-cellulose-ester membranes of 0.45 μm pore size. The membranes were transferred to tubes containing 10 ml peptone water. After the filter was suspended in the tube, serial dilutions of this were made (10⁻¹ to 10⁻⁴), and aliquots of 100 μl of each dilution were seeded onto dishes containing MacConkey agar. The dishes were incubated at 35°C for 48 hours and after incubation, the characteristic lactose negative colonies were selected.

Identification of the isolates of Acinetobacter spp.

The phenotypic identification of the isolates was made using Gram stain, the triple sugar iron (TSI) and oxidase biochemical tests. The genus was confirmed by amplifying a fragment of 16S rDNA, using the pair of nucleotide primers Acin16S (5′-CCT TGC GYT AAT AGA TGA GC-3′) and Acin16S (5′-GTA GCA ACC CTT TGT ACC GA-3′), which are specific for the genus Acinetobacter. This primer was designed using the sequence alignment available in GENBANK, of the 16S rRNA gene of 29 species of the genus Acinetobacter, using the program CLC FreeWorkbench 2.2. The amplification reactions were carried out in mixtures containing 3.5 mM MgCl₂, 0.2 mM of dNTPs, 1 μM of each primer, 1 U of Taq polymerase, 1 × of reaction buffer of Taq polymerase, and 100 ng of bacterial DNA in a final volume of 30 μl. We used an Eppendorf Mastercycler Personal thermocycler, with the following amplification conditions: an initial denaturation cycle at 95°C for 5 min, followed by 25 denaturation cycles at 95°C for 1 min, annealing at 52°C for 1 min, extension at 72°C for 1 min, and a final extension cycle at 72°C for 8 min. The species A. baumannii was identified by amplification of the bla_OXA-51 gene fragment. This gene is found exclusively in isolates of this species and can, therefore, be used to identify it.²⁶

Antimicrobial susceptibility test

The antimicrobial susceptibility profiles of the isolates of Acinetobacter spp. were determined using the disk-diffusion technique, according to the norms of the Clinical Laboratory Standards Institute (2009). These antimicrobials were used: amikacin (30 μg), gentamicin (10 μg), ciprofloxacin (5 μg), ceftazidime (30 μg), piperacillin-tazobactam (100 μg/10 μg), ticarcillin-clavulanic acid (75 μg/10 μg), imipenem (10 μg), meropenem (10 μg), cefepime (30 μg), and aztreonam (30 μg). Isolates that showed resistance to four or more classes of antimicrobials were considered multiresistant. The strain P. aeruginosa ATCC 27858 was used as a quality control in the susceptibility tests.

Phenotypic MBL detection test

For phenotypic triage of MBL production, the disk-approximation test was carried out, using the substrates imipenem and ceftazidime and the inhibitors EDTA and 2-mercaptopropionic acid (2-MPA).^2,16,34 The phenotypic test for production of MBL was also carried out to identify imipenem-resistant isolates, by the E-test-MBL method.³⁰

Extraction of DNA and detection of bla genes

The DNA of the isolates was obtained by the boiling method, according to Misbah et al.²⁰ The isolates that were resistant to imipenem, meropenem, and/or with a positive triage for production of MBL were submitted to polymerase chain reaction (PCR) reaction to assess the presence of the bla_IMP, bla_VIM, bla_SPM-1, bla_OXA-23like, bla_OXA-51like, bla_OXA-24like, and bla_OXA-58like genes. The oligonucleotide primers listed in Table 1 were used. The reactions were carried out in mixtures containing 1 × of Go Taq Master Mix (Promega), 1 μM of each primer, and 100 ng of bacterial DNA, in a final volume of 15 μl. The amplification conditions were as follows: an initial denaturation cycle at 94°C for 5 min, followed by 30 cycles of 94°C for 1 min, annealing temperature as in Table 1, extension at 72°C for 1 min, and a final extension at 72°C for 6 min. The PCR products were visualized in 1% agar gel, stained with ethidium bromide.

Table 1.

Primers Used in the Present Study for Detection of Resistant Genes of Classes B and D of Ambler

Gene	Sequence of oligonucleotide primer	Annealing temperature	Expected size (bp)	Reference
bla _IMPlike	F2 (5′ GGA ATA GAG TGG CTT AAY TCT C 3′)	53°	188	Ellington et al.⁹
	R2 (5′ CCA AAC YAC TAS GTT ATC T 3′)
bla _VIMlike	F3 (5′ GAT GGT GTT TGG TCG CAT A 3′)	52°	390	Ellington et al.⁹
	R3 (5′ CGA ATG CGC AGC ACC AG 3′)
bla _SPM	F4 (5′ 5′-AAA ATC TGG GTA CGC AAA CG3′)	52°	271	Ellington et al.⁹
	R4 (5′ACA TTA TCC GCT GGA ACA GG3′)
bla _OXA-23like	F1 (5′ GAT CGG ATT GGA GAA CCA GA3′)	51°	570	Woodford et al.³¹
	R1 (5′ ATT TCT GAC CGC ATT TCC AT 3′)
bla _OXA-51like	F1 (5′ TAA TGC TTT GAT CGG CCT TG3′)	53°	353	Woodford et al.³¹
	R1 (5′ TGG ATT GCA CTT CAT CTT GG 3′)
bla _OXA-24like	F1 (5′ GGT TAG TTG GCC CCC TTA AA 3′)	53°	246	Woodford et al.³¹
	R1 (5′AGT TGA GCG AAA AGG GGA TT3′)
bla _OXA-58like	F1 (5′AAG TAT TGG GGC TTG TGC TG 3′)	53°	599	Woodford et al.³¹
	R1 (5′CCC CTC TGC GCT CTA CAT AC 3′)

Enterobacterial repetitive intergenic consensus-PCR

The genetic similarity of the Acinetobacter junii isolates was determined by the enterobacterial repetitive intergenic consensus (ERIC)-PCR. The PCR reaction was performed in a final volume of 30 μl containing 8 μl of genomic DNA, 5.5 mM MgCl₂, 1.25 mM of each dNTP, 400 ng of each primer (ERIC1 and ERIC2), and 2 U of Taq DNA polymerase (Invitrogen). The amplification conditions were as follows: an initial denaturation cycle at 95°C for 7 minutes, followed by 30 cycles of 94°C for 1 minute, 51°C for 1 minute, and 72°C for 1 minute and 30 seconds, and a final extension at 72°C for 15 minutes. The PCR products were visualized in 2% agarose gel plus polymer Synergel (BioAmerica) in Tris-borate, stained with ethidium bromide 0.5 μg/ml.

Sequencing

PCR products of the 16S rDNA fragment from selected isolates were purified using the EZ-10 Spin Column PCR* Products Purification Kit and sequenced on a MEGAbace 1000 sequencer (GE), using the DYEnamic ET Terminator Kit. Sequences were analyzed using BLAST 2.0 (www.ncbi.nlm.nih.gov/BLAST) and submitted to GenBank (GenBank submission no GU289231, GU29535, and GU299536).

Results

We identified 303 isolates of Acinetobacter sp. in wastewater from the three hospitals: 105 isolates from the Hospital São Lucas, 97 from the Hospital Conceição, and 101 from the Hospital de Clínicas. The PCR for detection of the bla_OXA51 gene was carried out with all the isolates used in this study, and 56% showed a positive result. The bla_OXA51 gene is considered intrinsic to the species A. baumannii, and it can be used to identify it. In the Hospital São Lucas, 65% of the isolates were identified as A. baumannii. A similar result was found in the Hospital de Clínicas (66%), whereas the Hospital Conceição showed a smaller number of isolates of this species (37%).

In the Hospital de Clínicas, 43% of the isolates showed susceptibility to all the antimicrobials tested. The Hospital Conceição showed 22% susceptible isolates, whereas the Hospital São Lucas showed the largest number of susceptible isolates (67%) compared with the other hospitals (Table 2). In all three hospitals, the largest number of isolates were susceptible to one of the carbapenems: 95.14% of isolates were susceptible to imipenem, and 85.39% were susceptible to meropenem. The Hospital São Lucas had 21% multiresistant isolates; and the Hospital de Clínicas and the Hospital Conceição showed similar results, with 40% and 51.41% multiresistant isolates, respectively. The three hospitals showed higher percentages of resistance to the antimicrobials amikacin (39.27%), ceftazidime (40.26%), and ciprofloxacin (42.24%). In the Hospital Conceição, a high rate of resistance to gentamicin, with 57% resistant isolates, was observed. In the Hospital de Clínicas, 40% of the isolates showed resistance to Ticarcillin-clavulanic acid (Table 3). In the Hospital São Lucas, one isolate showed reduced susceptibility to all the antimicrobials tested; however, in the Hospital de Clínicas, it was two isolates, and no isolate in the Hospital Conceição.

Table 2.

Prevalence of Resistance to Multiple Antibiotics in Acinetobacter spp. Isolated from the Wastewater of Three Hospitals in Porto Alegre, Rio Grande do Sul, Brazil

No. of antibiotics	Hospital São Lucas % of isolates	Hospital Conceição % of isolates	Hospital de Clínicas % of isolates
Susceptible to all	67	22	43
Resistant to 1	9	6	7
Resistant to 2	6	12	9
Resistant to 3	1	6	2
Resistant to 4	7	11	2
Resistant to 5	6	4	3
Resistant to 6	7	11	29
Resistant to 7 or more	1	27	6

Table 3.

Percentage of Susceptibility to Antimicrobials of Acinetobacter spp. Isolated from Samples of Wastewater from Three Hospitals in Porto Alegre, Rio Grande do Sul, Brazil

	Hospital São Lucas (n = 105)			Hospital Conceição (n = 97)			Hospital de Clínicas (n = 101)
Antimicrobials	R	I	S	R	I	S	R	I	S
Amikacin	24	14	61	54	10	36	41	6	53
Aztreonam	13	50	36	40	29	31	49	32	20
Ceftazidime	23	11	66	52	7	41	47	15	39
Cefepime	11	11	77	36	7	57	39	7	54
Ciprofloxacin	18	2	80	69	3	28	41	2	57
Gentamicin	20	6	74	57	3	40	10	19	71
Imipenem	1	0	99	11	1	88	2	0	98
Meropenem	3	1	96	11	22	67	2	6	92
PIP	2	3	95	25	15	60	2	18	80
TIC	11	5	84	38	27	35	40	2	58

PIP, Piperacillin-Tazobactam; TIC, Ticarcillin-clavulanic acid; R, resistant; I, intermediate; S, susceptible.

In the phenotypic triage for MBL, production was carried out with all the isolates. Using the antimicrobial imipenem and the inhibitor EDTA, 3.81% of the isolates from the Hospital São Lucas were positive, 2.06% from the Hospital Conceição were positive, and there was no positive isolate from the Hospital de Clínicas. In the test using the antimicrobial ceftazidime and the inhibitor 2-MPA, a larger number of isolates was found in all the hospitals: In the Hospital São Lucas, 21% of the isolates showed a positive result in the test; in the Hospital Conceição, it was 14.43%; and in the Hospital de Clínicas, it was 14.85%. Only four isolates showed a positive result in both tests. All the isolates that showed resistance to imipenem were tested using E-test for detection of MBL; of the 35 isolates tested, 13 showed a positive result (Table 4).

Table 4.

Positive Results of the Phenotypic Metallo-β-Lactamases Tests and Detection of Metallo-β-Lactamases and Oxacilinases Genes Performed in Acinetobacter spp. Isolated from the Wastewater of Three Hospitals in Porto Alegre, Rio Grande do Sul, Brazil

				Detection of bla genes
	Phenotypic MBL tests			MBL		OXA
Hospitals	E-test (n = 35)^a	IPM/EDTA (n = 303)	CAZ/2-MPA (n = 303)	IMP^b (n = 77)	VIM^b (n = 77)	SPM^b (n = 77)	OXA-23^b (n = 77)	OXA-51^c (n = 303)	OXA-24^b (n = 77)	OXA-58^b (n = 77)
Clínicas (n = 101)	2	0	15	0	0	0	2	67	0	0
São Lucas (n = 105)	0	4	22	0	0	0	1	68	0	0
Conceição (n = 97)	11	2	14	0	0	0	0	36	0	0

Test performed only with imipenem resistant isolates.

Test performed with carbapenems resistant isolates or with a positive result in one of the triage test.

Test performed with all isolates.

MBL, metallo-β-lactamases.

The amplification of the fragments of the bla_IMP, bla_VIM, and bla_SPM-1 genes was carried out with 77 isolates that showed reduced susceptibility to carbapenems, that is, those isolates which appeared resistant or intermediate in the disk-diffusion antibiogram. None of the isolates carried genes for production of the MBLs tested. The same isolates were tested for the bla_OXA23like, bla_OXA24like, and bla_OXA58like genes. As mentioned earlier, 171 isolates were positive to bla_OXA-51; only three isolates were positive to bla_OXA23like; and none of the isolates were positive to bla_OXA24like and bla_OXA58like genes. One of the isolates positive to bla_OXA23like was identified in wastewater from the Hospital São Lucas, and two isolates positive to bla_OXA23like was identified in wastewater from the Hospital de Clínicas (Table 4). The three isolates showed loss of susceptibility to all the antibiotics tested. These isolates also carried the bla_OXA-51 gene and were, therefore, identified as A. baumannii.

When we compared the resistance profile of A. baumannii isolates with other Acinetobacter sp., we observed a similar pattern in the overall distribution of the resistance, but the first ones were more resistant for the majority of tested antibiotics (Fig. 1). However, this result was not observed among the carbapenems and gentamicin due to 11 isolates from Hospital Conceição that were not identified as A. baumannii. They had the same resistance profile and were resistant to gentamicin, meropenem, and imipenem; were positive in the E-test MBL; and were negative for the presence of bla_IMP, bla_VIM, bla_SPM-1, bla_OXA-51, bla_OXA24like, bla_OXA58like, and bla_OXA-23 genes. These 11 isolates shared many phenotypic characteristics were isolated from the same sampling site, and showed two distinct ERIC-PCR patterns (Supplementary Fig. S1 available online at www.liebertonline.com/mdr). Three of these 11 isolates, with different ERIC-PCR pattern, had their 16S rDNA fragment sequenced. They all presented the same sequence with 98% identity with A. junii 16S rRNA sequences from Genbank.

FIG. 1.

Comparison among the resistance profile of Acinetobacter baumanii and Acinetobacter spp. isolated from wastewater samples of Porto Alegre/RS hospitals. AMI, amikacin; AZT, aztreonam; CAZ, ceftazidime; CPM, cefepime; CIP, ciprofloxacin; GEN, gentamicin; IMP, imipenem; MER, meropenem, PIP, piperacillin-tazobactam; TIC, ticarcillin-clavulanic acid.

Discussion

Few studies have been carried out to determine antimicrobial-susceptibility profiles in bacterial isolates obtained from samples of hospital wastewater. Guardabassi et al.¹² determined the antimicrobial profile of isolates of Acinetobacter sp. from hospital wastewater and a pharmaceutical company, and they observed that the incidence of resistance of the isolates from the wastewater, compared with the clinical samples, was generally lower. However, in this study, few antimicrobials were tested, and the carbapenems, which are the antimicrobials of choice for treatment of infections caused by Acinetobacter spp., were not tested. Even so, it was possible to observe the presence of 2.2% isolates that were resistant to three or more antimicrobials of the six tested. Yang et al.,³³ in a study comparing the antimicrobial-resistance profile between clinical isolates and isolates from hospital wastewater in Taiwan, concluded that isolates from wastewater showed high rates of microbial resistance, as did the clinical isolates from the same hospital. In our studies, we found 30% multiresistant isolates in the three hospitals wastewaters analyzed; this high percentage is indicative of the contribution by the hospital environment. In the Hospital Conceição and the Hospital de Clínicas, where the collections were made at different points inside each hospital, the wastewater from the laundry yielded the largest number of isolates, 50 and 56, respectively. In another study of our group, it was observed that more multiresistant isolates were found at these points than at the other points analyzed.¹⁰

The triage test for production of MBLs was shown to be inefficient for the isolates of Acinetobacter spp. tested in this study: Several isolates gave positive results but did not carry genes for the production of these enzymes. Most of the isolates that were positive in these tests were bla_OXA-23 and bla_OXA-51 positive, with exception of the A. junii isolates that were positive for the E-test and the CAZ/2-MPA test and negative to all tested genes. Comparative studies among phenotypic tests for the detection of MBLs were carried out by Yan et al.,³² demonstrating that these tests, although very good for other bacteria genus, are inefficient for isolates of the genus Acinetobacter; and we suggest that other tests be developed.

The OXA-51-type enzyme was first found in a clinical isolate in Argentina, but several studies have now revealed that this gene is present in the genus Acinetobacter in several continents.²⁸ The OXA-51-type enzyme group includes a large number of variants with the numbers 64, 65, 66, 67, 68, 69, 70, 71, 75, 76, 77, 83, 84, 86, 87, 88, 89, 91, 92, 94, and 95. These genes are chromosomal, occur naturally in isolates of A. baumannii, and do not appear in other species of this genus.²⁷ For this reason, many studies have used the bla_OXA-51like gene to identify this species, which is the most common in nosocomial infections caused by the genus Acinetobacter. The presence of this gene is not necessarily related to resistance to carbapenems, because it depends on the ISAbaI insertion sequence, which when located above the bla_OXA-51 functions as a promotor of the expression, contributing to increased expression of resistance to carbapenems.¹⁹ According to a study by Vahaboglu et al.,²⁸ 77.8% of the clinical isolates analyzed carried the bla_OXA-51 gene, belonging to the species A. baumannii; and these isolates also showed high rates of antimicrobial resistance.

In this study, more than half of the isolates carried the bla_OXA-51like gene, indicating a high prevalence of isolates of A. baumannii in the hospital wastewater; this is the species that is most often associated with hospital-acquired infections. Studies have shown that this species, together with Acinetobacter calcoaceticus, Acinetobacter genomospecies 3, and 13TU, which form the so-called A. baumannii-calcoaceticus complex, show the highest rates of antimicrobial resistance. In this study, of the 44 isolates that showed decreased susceptibility to carbapenems, 20 were identified as A. baumannii; this resistance may be associated with the presence of the bla_OXA-51like gene, as they present the insertion sequence ISAbaI, located upstream the gene. Bratu et al.⁶ reported that the OXA-51-lactamase possesses only slow hydrolytic activity against imipenem. A larger number of multiresistant isolates belonging to the species A. baumannii was also observed when comparing with isolates identified as Acinetobacter spp. Bratu et al.⁶ showed that in A. baumannii, reduced susceptibility to cephalosporins can be associated with efflux pump systems (AdeABC); on the other hand, it is not an important contributor to aminoglycoside and carbapenem resistance, the first one normally required the presence of an aminoglycoside-modifying enzyme. These efflux pump systems also contribute to the intrinsic resistance to many antimicrobial agents, dyes, detergents, and chemicals that are often found in hospital wastewater and might explain the high resistance rates among the strains isolated in our study. Takagi et al.²⁵ observed that carbapenem resistant phenotype in A. baumanii isolates, recovered from eight patients in an intensive care unit in Brazil, was related to loss of a 22 kDa outer membrane protein and the presence of bla_OXA-51-like β-lactamase genes. In other Acinetobacter spp. isolates, such as the A. junii, these nonenzymatic mechanisms, including the lack of outer membrane proteins as the OmpA-like porin, should be also related to the resistance observed; and future studies should be done to investigate it.

We had also identified two clones of the species A. junii, with similar phenotypic characteristics, among the multiresistant isolates, that were responsible for the high resistance to gentamicin and, in a less rate, to carbapenems. In the last few years, this species has been described as a rare opportunistic pathogen found in the environment,^5,15 but Hung et al.¹³ reported it as an emerging pathogen that mainly affects patients with malignancies or invasive procedures and who have had prior antimicrobial therapy. They also observed high rates of resistance to gentamicin but low rates of resistance to carbapenems among the isolates, and they emphasized that the increasing antimicrobial resistance among hospital acquired isolates of A. junii should be monitored.

According to data from SENTRY, the bla_OXA-23 gene was the one most often found in studies of clinical samples in medical centers in Asia, corresponding to 95% of the Class D genes.¹⁸ In the Military Medical Academy in Bulgaria, clonal dissemination of isolates of Acinetobacter sp. with the bla_OXA-23 gene was also observed.²⁴ Dalla-Costa et al.,⁸ in a study in two hospitals in Curitiba, Brazil, found multiresistant OXA-23-producing isolates. Carvalho et al.⁷ observed the dissemination of clones of multiresistant strains of A. baumannii carriers of the bla_OXA-23like gene in eight hospitals in Rio de Janeiro, Brazil. Many studies with clinical samples, in various parts of the world, have identified isolates that produce this enzyme. In the hospitals analyzed in this study, strains with the bla_OXA-23like gene were identified among clinical isolates (data not shown) as well as in isolates from the wastewater from these hospitals. The presence of multiresistant OXA-23-producing strains of A. baumannii in wastewater from these hospitals indicates that the pathogenic strains which circulate in the hospital environment can reach the wastewater and can, therefore, enter local waterbodies.

This is the first study to evaluate the dissemination of these resistance genes in hospital wastewater. Although the carbapenem resistance has been attributed to the association of the promoter sequence ISAba1, the results indicate that multiresistant isolates of A. baumannii carrying resistance genes such as bla_OXA-51like and bla_OXA-23like are being released into the environment through the wastewater of the hospitals analyzed. We also found multiresistant A. junii isolates in the hospital wastewater and since they were all negative for the tested genes, they might have another resistance mechanism that should be investigated in future. Therefore, the hospital wastewater may be a factor in the development and dissemination of multiresistant bacteria and resistance genes into the environment, making the local waterbodies potential reservoirs of bacterial resistance.

Footnotes

Acknowledgments

Our thanks to the Hospital Infection Control Committee from Hospital de Clínicas de Porto Alegre, Hospital São Lucas, and Hospital Conceição, who kindly authorized the collection of the wastewater samples and the publishing of the results, and to Dr. Ana Cristina Gales (Laboratório Alerta and Laboratório Especial de Microbiologia Clínica, Division of Infectious Diseases, Universidade Federal de São Paulo), who kindly provided the strains used as positive controls. This study had the financial support of CAPES-PROF.

Disclosure Statement

No competing financial interests exist.

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