Multilocus Sequence Typing of Carbapenem-Resistant Acinetobacter baumannii Isolates Harboring bla OXA-23 and bla IMP in Cattle from Punjab,Pakistan

Abstract

Acinetobacter baumannii is a notorious bacterial pathogen that can cause an array of nosocomial infections in clinical settings. However, the data from the veterinary settings is limited and especially in Pakistan, no such study is conducted so far. To investigate the prevalence, antimicrobial resistance, and distribution of specific sequence types of A. baumannii in cattle, a total of 1,960 samples were collected from cattle over 18 months from Punjab, Pakistan. The isolates obtained were identified using the API20NE system and confirmed through PCR. The isolated A. baumannii isolates were further screened for antimicrobial susceptibility and the presence of resistance genes. Multilocus sequence typing was carried out to characterize the carbapenem-resistant A. baumannii (CRAB) isolates. Results revealed an overall prevalence of A. baumannii at 3.31% (65/1,960) with a higher prevalence of 7.38% (54/731) in dairy cattle compared to beef cattle at 4.41% (11/249). Among 65 A. baumannii isolates, 27.7% (18/65) were CRAB. All CRAB isolates harbor class D β-lactamases genes bla_OXA-23 and bla_OXA-51, whereas 94.4% (17/18) CRAB isolates carried class B β-lactamases gene bla_IMP, and only one isolate had bla_NDM-1 gene. The commonly found sequence types for CRAB isolates were ST2 and ST642 corresponding to 10 and 05 isolates, respectively. The presence of CRAB in cattle indicates an alarming situation that necessitates an urgent and efficient surveillance system to limit the transmission of CRAB among the cattle population and its possible transmission to humans and the environment.

Introduction

The emergence of carbapenem-resistant Acinetobacter baumannii (CRAB) is a public health care problem throughout the world. Many species of genus Acinetobacter are opportunistic and are associated with nosocomial infections; however, the A. baumannii is considered the foremost important due to its involvement in clinical outbreaks and increasing resistance to multiple antibiotics, especially carbapenems.¹

Carbapenems are considered as the drug of last resort for the therapeutic management of infections caused by bacterial pathogens; therefore, the widespread resistance among the A. baumannii against carbapenems is a great threat to public health that ultimately leads to an increased rate of infections, prolonged hospitalization, and higher mortality rates.² The presence of oxacillinases such as bla_OXA-23, bla_OXA-58, and bla_OXA-24 in Acinetobacter species are mainly conferring carbapenem resistance to the A. baumannii isolates. However, these are considered weak carbapenemases and the presence of insertion sequences that is, ISAba1 upstream to these oxacillinases gene results in increased expression of these genes leading to carbapenem-resistant phenotypes in A. baumannii.¹

The Class B β-lactamase with stronger carbapenem hydrolyzing activity includes the bla_IMP, bla_VIM, and bla_NDM-1, which play a significant role in the development of resistance against carbapenems. Although these class B enzymes, especially the bla_NDM-1, have been reported in several Enterobacteriaceae species, few reports have indicated the presence of these enzymes in A. baumannii isolated from humans in several countries.³

The A. baumannii is also responsible for causing disease in veterinary settings.^4–6 Moreover, several studies revealed the presence of A. baumannii in companion animals.^4,5,7 A. baumannii from the nosocomial sources mainly belongs to eight international clones (ICs), among which the most prevalent are IC2 and IC1, which are also known as sequence types ST2 and ST1 according to Pasteur scheme.⁸ The studies have reported the presence of CRAB isolates belonging to ST1 and ST2 in companion animals.^5,7 The studies have reported that the A. baumannii isolated from bovine mainly corresponded to new sequence types (STs), whereas the dominant STs were rarely detected.⁶

In developing countries like Pakistan, the data related to the prevalence, antibiotic-resistant features, resistant determinants, and genetic diversity of A. baumannii are not available from the veterinary settings. So, the current study is designed to determine the prevalence, antimicrobial resistance, and distribution of sequence types of A. baumannii in the cattle population of Punjab, Pakistan.

Materials and Methods

The study got prior approval from the institutional review board of the Government College University Faisalabad, Pakistan to isolate the bacterial strains and collect the data for this study.

Isolation and identification of bacteria

A total of 980 cattle were included in this study. From each cattle, the nasopharyngeal swabs (NS), n = 980, and rectal swabs (RS), n = 980, were collected over a period of 18 months from various districts of Punjab province, Pakistan. The NS and RS were initially cultured on blood and MacConkey Agar (Oxoid, United Kingdom) to observe colony morphology followed by Gram staining. The isolates obtained were further processed for identification using the API20NE (bioMérieux, Marcy l'Etoile France) followed by VITEK^® 2 system (bioMérieux, Marcy l'Etoile France). Further confirmation was done by PCR by amplifying the internal transcribed spacer region (208 bp) and 425 bp fragments of the recA gene.¹ A questionnaire was used to collect data regarding age, sex, season, type of feeding, previous use of antibiotics, and so on.

Antimicrobial susceptibility testing

The antimicrobial susceptibility testing was performed by Kirby Bauer disc-diffusion assay followed by the determination of minimum inhibitory concentration (MIC) using broth microdilution assay. The A. baumannii isolates were cultured on Mueller Hinton agar (Oxoid) for susceptibility testing as per CLSI 2020 guidelines using various antimicrobial discs (Oxoid); Ampicillin-sulbactam (SAM), Piperacillin-tazobactam (TZP), Ceftazidime (CAZ), Cefepime (FEP), Cefotaxime (CTX), Ceftriaxone (CRO), Imipenem (IMP), Meropenem (MEM), Gentamicin (CN), Tobramycin (TOB), Amikacin (AK), Doxycycline (DO), ciprofloxacin (CIP), and Trimethoprim-sulfamethoxazole (SXT).

The MIC provides a quantitative susceptibility pattern of bacterial isolates against certain antimicrobial agents. For the micro broth dilution assay, Mueller Hinton broth (Oxoid) was used to determine the MIC of the following drugs with the following MIC breakpoints; Ampicillin-sulbactam (≥32/16 μg/mL), Piperacillin-tazobactam (≥128/4 μg/mL), Ceftazidime (≥32 μg/mL), Cefepime (≥32 μg/mL), Cefotaxime (≥64 μg/mL), Ceftriaxone (≥64 μg/mL), Imipenem (≥8 μg/mL), Meropenem (≥8 μg/mL), Gentamicin (≥16 μg/mL), Tobramycin (≥16 μg/mL), Amikacin (≥64 μg/mL), Doxycycline (≥16 μg/mL), Ciprofloxacin (≥4 μg/mL), Trimethoprim-sulfamethoxazole (≥4/76 μg/mL), and Colistin and Polymyxin B (≥4 μg/mL) according to Clinical and Laboratory Standards Institute guidelines (2020). For tigecycline susceptibility, Food and Drug Authority criteria for Enterobacteriaceae was followed, that is, the isolates with a MIC ≥8 μg/mL were considered as resistant and for quality control, Escherichia coli (ATCC^® strains 25,922 and 35,218) and Pseudomonas aeruginosa ATCC strain 27,853.⁹

Detection of β-lactamases genes

The isolated A. baumannii were screened for detection of various carbapenemases genes using specific primers (Supplementary Table S1). The class B β-lactamases genes include bla_IMP, bla_VIM, bla_GIM, bla_NDM-1, and bla_SIM. The class D β-lactamases include bla_OXA23-like, bla_OXA24-like, bla_OXA51, and bla_OXA58-like. The screening of ISAba1 upstream to the class D carbapenemases was also carried out as described previously.¹ The amplified product was run on electrophoresis using 1.2% agarose gel containing 0.5 μg/mL ethidium bromide. The amplicons were purified and shipped for sequencing by the Sanger sequencing method at Macrogen™ (Seoul, South Korea).

Multilocus sequence typing

To find out genetic diversity among A. baumannii isolates, multilocus sequence typing (MLST) was performed following the Pasteur Scheme. Amplification of seven housekeeping genes (cpn60, fusA, gltA, pyrG, recA, rplB, and rpoB) was done followed by sequencing. For sequence analysis, the online tools such as PubMLST database were used. Genetic relatedness among STs was accessed following stringent criteria for having the same alleles for at least six of the seven loci.¹⁰

Statistical analysis

The chi-square test and t-test were used to compare categorical variables and to compare the mean values, respectively.

Results

Prevalence of A. baumannii in cattle

The overall prevalence of A. baumannii was 3.31% (65/1,960) with a higher prevalence in dairy cattle 7.38% (54/731) than beef cattle 4.41% (11/249). The prevalence rate was significantly higher 6.02% (59/980) in NS compared to 0.61% (6/980) in RS (Table 1).

Table 1.

Characteristics of Animals Included in the Study

Variables	Categories	No. of specimens	No. of specimens harboring Acinetobacter baumannii	No. of specimensdo not harbor A. baumannii	Total %	Statistical analysis
Age of animals	0–19 months	249	11	238	4.41	χ² = 2.64 df = 1 p = 0.10
Age of animals	20–58 months	731	54	677	7.38	χ² = 2.64 df = 1 p = 0.10
Dairy and beef cattle	Dairy cattle	731	54	677	7.38	χ² = 2.64 df = 1 p = 0.10
Dairy and beef cattle	Beef cattle	249	11	238	4.41	χ² = 2.64 df = 1 p = 0.10
Feeding of corn silage	Dairy cattle	700	54	643	7.71	χ² = 0.40 df = 1 p = 0.52
Feeding of corn silage	Beef cattle	197	11	186	5.58	χ² = 0.40 df = 1 p = 0.52
Sex of the animal	Female	731	54	677	7.38	χ² = 2.64 df = 1 p = 0.10
Sex of the animal	Male	249	11	238	4.41	χ² = 2.64 df = 1 p = 0.10
Systemic use of third generation cephalosporins	Dairy cattle	685	53	632	7.73	χ² = 0.77 df = 1 p = 0.37
Systemic use of third generation cephalosporins	Beef cattle	173	10	163	5.75	χ² = 0.77 df = 1 p = 0.37
Seasonal distribution	January–April	334	5	329	1.49	χ² = 5.64 df = 2 p = 0.05^a
	May–August	1,166	47	1,119	4.04
	September–December	464	13	447	2.80
Specimens	Nasal swabs	980	59	921	6.02	t-test values p = 0.0015^a
Specimens	Rectal swabs	980	08	972	0.81	t-test values p = 0.0015^a

Statistical significance.

The different characteristics of cattle like age, sex, breed, type of feeding, season, farm size, previous use of antibiotics are shown in Table 1. The overall occurrence of A. baumannii was higher at 4.04% (47/1,162) from May to August followed by 2.80% (13/464) during September to December and 1.49% (5/334) during January to April, respectively. The seasonal difference was statistically significant (p-value <0.05). The A. baumannii isolates were more common among female cattle population, 7.38% (54/731), compared to male, 4.41% (11/249).

Antimicrobial susceptibility

Out of 65 A. baumannii isolates, 33 (51%) exhibited resistance against Ampicillin-sulbactam, 34 (52%) against piperacillin-tazobactam, 18 (27.7%) against ceftazidime, cefepime, cefotaxime, ceftriaxone, imipenem, and meropenem, 43 (66%) to gentamicin and amikacin, 33 (51%) tobramycin, whereas 30 (46%), 33 (51%), and 39 (60%) were resistant to doxycycline, ciprofloxacin, and trimethoprim-sulfamethoxazole, respectively. All A. baumannii isolates were susceptible to polymyxin B, colistin, and tigecycline. The distribution of MICs for A. baumannii isolates against the antimicrobial agents is mentioned in Supplementary Table S2.

Among the 65 A. baumannii isolates, 27.7% (18/65) were resistant to carbapenems. Moreover, CRAB isolates showed higher resistance, 100% (18/18) against ampicillin-sulbactam, piperacillin-tazobactam, ceftazidime, cefepime, cefotaxime, ceftriaxone, imipenem, meropenem, gentamicin, tobramycin, amikacin, trimethoprim-sulfamethoxazole, and ciprofloxacin. All the CRAB isolates were susceptible to polymyxin B, colistin, and tigecycline. The distribution of MICs for the CRAB isolates against antimicrobial agents is mentioned in Supplementary Table S3.

Detection of β-lactamases genes

All the 65 A. baumannii isolates were positive for the blaOXA-51 gene. While all 18 CRAB isolates harbored both class D β-lactamases genes, that is, bla_OXA-23 and bla_OXA-51. All these 18 isolates with a carbapenem-resistant phenotypes were harboring ISAba1 upstream to the bla_OXA-51 as well as bla_OXA-23. Among the class B enzymes, the bla_IMP gene was present in 94.4% (17/18) isolates and one isolate was having the bla_NDM-1 gene.

Multilocus sequence typing

The analysis of MLST has shown that a total of 5 STs were found for the 18 CRAB isolates as shown in Supplementary Table S4. The commonly found sequence types for CRAB were ST2 55.5% (n = 10), ST642 27.8% (n = 05), and ST600 and ST889 5.55% (n = 01).

Discussion

A. baumannii is an emerging problem in clinical settings because of its involvement in severe nosocomial infections leading to increase mortality and morbidity. A. baumannii strains have acquired resistance to most available antibiotics, especially carbapenems, therefore, included in the top priority pathogens list by World Health Organization.¹¹ The data regarding the prevalence and antimicrobial profiling of A. baumannii are quite limited in veterinary settings in the world, whereas no study is available from Pakistan. A few studies were conducted to find out the prevalence of Acinetobacter species in cattle from Poland, Germany, and France.^12,13 The present study revealed the CRAB in beef and dairy cattle in Punjab, Pakistan, for the first time.

The overall prevalence of A. baumannii was 3.31% (65/1,960) with a higher prevalence in dairy cattle 7.38% (54/731) than beef cattle 4.41% (11/249). Among 65 A. baumannii isolates, 27.7% (18/65) were found CRAB with an overall recovery rate of 0.009 (18/1,960). Different studies have reported a variable recovery rate of A. baumannii from the farm animals. A study reported a recovery rate of CRAB as 0.6% from the feces of dairy cattle.¹³ The study from a single farm reported an isolation rate of 83% from the oral swabs of cattle in the Reunion Islands; however, it included only six animals.¹⁴

The overall occurrence of A. baumannii was higher at 4.04% (47/1,162) from May to August followed by 2.80% (13/464) during September to December and 1.49% (5/334) during January to April, respectively. The A. baumannii isolates were more common among female cattle population, 7.38% (54/731), compared to male, 4.41% (11/249). The data from clinical settings have reported the seasonal variations in the isolation of Acinetobacter species. A study has reported that a 5.6°C increase in the temperature can increase the infection rates by 17% for A. baumannii.¹⁵

The prevalence rate was significantly higher by 6.02% (59/980) in NS compared to 0.61% (6/980) in RS. The noses of cattle have a higher prevalence of A. baumannii that might be due to the acquisition of bacterium through inhaling of the feed particles or contaminated air from the surroundings. In fact, the airborne transmission of A. baumannii in the hospital environment is reported in some studies.^16,17 Furthermore, the contamination of air with Acinetobacter species in animal sheds has been reported.⁶ The Acinetobacter isolates have been recovered from certain plants, including maize as well as maize silage, which are being used as feed in the cattle farms.^18,19

The antimicrobial susceptibility data showed that among 18/65, 27.7% A. baumannii isolates were CRAB. The percentage of CRAB among the A. baumannii is different in cattle isolates compared to the reported strains found in the humans and companion animals, where carbapenem resistant seems to be a usual phenotype.^1,7,9,10 The antimicrobial resistance profile of the majority of A. baumannii isolates from cattle resembles the environmental strains and isolates from wildlife animals.²⁰

All 100% (18/18) CRAB isolates harbored class D β-lactamases genes, that is, bla_OXA-23, and bla_OXA-51, whereas 94.4% (17/18) carried class B β-lactamases gene that is, bla_IMP, and only one isolate had bla_NDM-1 gene. The resistance to carbapenems is predominantly mediated by the Class D beta-lactamases. For example, the oxacillinase blaOXA-51 is intrinsically present in the A. baumannii species and can confer resistance when overexpressed. Further, the acquisition of the other oxacillinases notably bla_OXA-23, bla_OXA-24, bla_OXA-58, bla_OXA-143, and bla_OXA-235 also confer carbapenem resistance. The insertion sequences can upregulate these oxacillinases genes if present upstream to these genes, for example, ISAba1.²¹ In the present study, all the CRAB isolates were harboring the acquired oxacillinase, that is, bla_OXA-23. This was evident from our previous studies in clinical settings, which showed the presence of the bla_OXA-23 gene in almost all CRAB isolates.^1,10

However, these studies have shown that the Metallo-beta-lactamases (MBLs) such as bla_IMP, bla_VIM, blaGIM, and blaSIM were not found in any of the isolates and blaNDM have been found in only seven clinical isolates. Therefore, this is the first report regarding the presence of MBLs, especially the blaIMP, among A. baumannii isolates from Pakistan. The MBLs producing A. baumannii strains are frequently reported from neighboring countries such as Iran.^22,23 However, these reports are from clinical settings and the presence of such carbapenem resistance mechanisms in veterinary stings is quite alarming.

The CRAB isolates were examined by MLST, five STs were found, including one new ST. The commonly found sequence types in CRAB were ST2 55.5% (n = 10), ST642 27.8% (n = 05), and ST600 and ST889 5.55% (n = 01). The information about the STs of A. baumannii strains from the animal carriage is quite little. It is noteworthy that ST2 is a prevalent sequence type in A. baumannii strains isolated from cattle. ST2 has been widely reported in human infections due to CRAB in different continents.^24–27

Although the results indicate the likelihood of human contamination, however, the possibility of an animal reservoir of this dominant clone cannot be denied. In contrast to our findings, the previous studies of MLST from Lebanon, France, and Germany reported huge diversity of A. baumannii strains in the cattle.^14,28 The presence of the dominant human lineages, that is, ST2 among the cattle field isolates, also indicates the transmission of these strains from the cattle population to humans.

Therefore, large-scale studies involving different regions should be conducted on a mass scale for characterization of the CRAB and for understanding the molecular epidemiology of the predominant clone in the region.

Conclusion

This is the first study that has reported the presence of A. baumannii in bovine fields in Pakistan. Moreover, the molecular mechanisms of carbapenem resistance in the CRAB isolates and their MLST types have been established. The results have shown that A. baumannii is an important species in cattle that should be taken into consideration. Compared to the human strains, the population of A. baumannii in bovine is still susceptible to carbapenems; however, it is diverse in terms of carbapenem-resistant mechanisms with the presence of MBLs that is frightening. Moreover, the strains are phylogenetically similar to the clinical isolates and are a potential threat; especially, these strains enter clinical settings and acquire the resistance genes.

It is important to understand the origin of bovine strains and the fact that whether these strains transiently colonize the animals for a longer time. The results are of great concern as the presence of CRAB in the cattle and the diverse carbapenem resistance mechanisms are not systematically investigated so far. Therefore, further large-scale studies are needed to establish the link between the transmission of such strains between the humans, environment, and animals using the one health approach.

Footnotes

Authors' Contributions

M.A.: conceptualization (equal), methodology (lead), investigation (lead), and writing—original draft (equal). M.H.R.: conceptualization (equal), supervision (lead), writing—original draft (equal), and writing—review and editing (equal). I.K.: conceptualization (supporting), funding acquisition (lead), and resources (equal). M.K.: conceptualization (supporting), formal analysis (equal), visualization (lead), and writing—review and editing (lead). B.A.: conceptualization (supporting), formal analysis (equal), and writing—review and editing (equal).

Disclosure Statement

No competing financial interests exist.

Funding Information

This research work was kindly supported by the German Federal Foreign Office under the project entitled “Lab networking under biosafety and biosecurity aspects in Pakistan”.

Supplementary Material

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