Outbreak of Multidrug-resistant Acinetobacter baumannii in a Tertiary Health Center from Northwestern Nigeria

Abstract

Background:

In spite of its global notoriety and WHO alarm, Acinetobacter baumannii is still an understudied critical-priority pathobiont in Nigeria. We characterized its antimicrobial susceptibility profile and resistance genes during an outbreak.

Materials and Methods:

This cross-sectional study involved collection of patients’ urine samples and swabs from unit staff’s hands and ward environments for the identification of A. baumannii strains using standard morphologic and biochemical methods. The disk diffusion method was used to assess the antimicrobial susceptibility profile of the isolates with the production of extended-spectrum beta-lactamases (ESBLs) confirmed by the combined disk test screening method. Characterization of the resistance genes of the ESBL producers was carried out using polymerase chain reaction polymerase chain reaction technique.

Results:

A total of eight (six clinical and two nonclinical) A. baumannii isolates were identified. The overall isolate susceptibility and resistance rates to all the antimicrobial agents was 56.3% (27/48) and 35.4% (17/48), respectively. Similarly, all (8/8; 100.00%) isolates were susceptible to meropenem and 75.0% (6/8) to ampicillin-sulbactam while 62.5% (5/8) were resistant to trimethoprim-sulfamethoxazole and 50.0% (4/8) to each of ciprofloxacin and ceftazidime. In addition, 37.5% (3/8) of the isolates were multidrug resistant (MDR) with nonclinical isolates exhibiting more antimicrobial resistance than their clinical counterparts (9/12%–75.0% vs. 8/36%–22.2%). Phenotypic detection and molecular characterization revealed three ESBL-producing isolates that each harbored bla_SHV and bla_TEM genes with bla_CTX-M gene being absent.

Conclusion:

MDR strains of A. baumannii harboring bla_SHV and bla_TEM genes were recovered from clinical and environmental sources during the outbreak, which was contained with preventive measures recommended.

INTRODUCTION

Reversal to the former epidemiological paradigm, where infectious diseases were the leading cause of mortality and disability globally, is being promoted by critical-priority pathogens like Acinetobacter baumannii. A. baumannii, the most virulent of the over 70 Acinetobacter spp. described; is a pleomorphic, biofilm-forming pathobiont belonging to the moraxellaceae family and responsible for significant social and economic burdens worldwide due to its propensity to cause life-threatening nosocomial infections/diseases (like pneumonia, meningitis, bloodstream, skin, wound and urinary tract infections), widespread multidrug-resistant (MDR) profile, tenacious survival tendencies, global endemicity, and attendant higher healthcare costs.[1,2] Globally, A. baumannii disproportionately contributes to the one million antimicrobial resistance (AMR)-attributable deaths in 2019 (Western sub-Saharan African countries like Nigeria were the worst hit), projected to wreak even more havoc by contributing to 10 million deaths annually by 2050 (surpassing cancer deaths) and increasing global healthcare costs to 100 trillion USD per year by 2050 if the situation is not urgently addressed.[3]

Generally, prevalence of A. baumannii vary according to geographic contexts, hospital units (intensive care unit [ICU] vs. other unit), patient’s body site (those with high fluid content-cerebrospinal fluid, peritoneal fluid, respiratory and urinary tracts; contain more), disease severity status (critically ill vs. not critically ill) and immunity status (immunocompromised vs. immunocompetent).[1,4,5] Since the first case of MDR A. baumannii clinical isolates was published in 2002 in Nigeria, its national prevalence has risen to 9.2% with 3.1% for Northwestern Nigeria.[5] However, information on community-acquired (from farm/food animals and environment) A. baumannii infections in Nigeria is even scantier.[3,5]

Due to its ubiquitous nature, infectious disease specialists have to constantly determine for each reported case of A. baumannii whether it is colonization or infection or even an outbreak as colonization often precedes infection/outbreak and only infection/outbreak should be treated.[6] MDR (especially carbapenem-resistant or plasmid-/integron-containing) strains of A. baumannii are responsible for most outbreaks in hospital settings in Nigeria[6] and other parts of the world.[2,4,7] MDR A. baumannii (MDRAB) causes outbreaks due to its remarkable resistance to desiccation; unrivalled genomic plasticity to acquire resistance genes to biocides and the last-resort antimicrobial agents (such as colistin and tigecycline/minocycline) through mobile genetic elements (like insertion sequences, transposons, integrons and plasmids); survival in harsh hospital environments (thus contaminating hospital surfaces and equipment, and medical/monitoring devices) and transfer/spread through contact among persons (healthcare personnel, patients), or from persons-animals (livestocks and pets) and-environment interactions.[3,4,7,8] Not surprisingly, A. baumannii was one of the two most frequently reported pathogens globally in the AMR surveillance systems in 2021;[3] as its rate of MDR strains quadruples that of other major nosocomial pathogens.[7]

In line with the mandates of the Global Action Plan, Global AMR and Use Surveillance System (GLASS), and National Action Plan; and to bridge the knowledge gap occasioned by the paucity of data on A. baumannii in Nigeria; we report the first published work on outbreak of the GLASS pathogen in Sokoto, Nigeria.

MATERIALS AND METHODS

Study area

The study was conducted at Usmanu Danfodiyo University Teaching Hospital (UDUTH), Sokoto, Nigeria, and involved the departments of Medical Microbiology and Surgery (urology unit).

Study design and population

This was a descriptive cross-sectional study comprising all patients that were admitted at urology unit in June 2020-period of the outbreak. They were identified via routine inter-departmental consult between the clinical microbiologists and the urologic surgeons.

Case definition and data collection

We defined a case as any patient from urology ward of UDUTH, Sokoto, Nigeria, from whose specimen isolates of A. baumannii resistant to at least three different classes of antimicrobial agents was detected. The cases were then traced back from the laboratory to the ward and their case files/folders were reviewed. Samples were retaken where necessary and the study was completed within a month.

Consequently, a MDR isolate is resistant to at least one agent in at least three antimicrobial classes.[9]

Sample collection

Urine samples were collected for microscopy, culture and sensitivity (MCS). Swabs were collected from the hands of staff members in the units, ward environment (including equipment/instrument), toilet sinks and floor of the urology ward and also subjected to MCS. Consequently, a total of 23 (7 from unit personnel and 16 from admitted patients) clinical samples and 55 environmental samples were collected.

Verbal and written consents of the nonliterate and literate study participants were, respectively, sought and obtained.

Sample processing

Urine samples were cultured on Cysteine Lactose Electrolyte Deficient agar. Swabs were cultured on blood agar and MacConkey agar. These were then incubated aerobically for 24 h under ambient air at 37°C.

Identification of isolates

Isolates with suspected colonial morphology of Acinetobacter spp. were subjected to Gram staining according to standard methods. The Oxoid Microbact™ Gram-negative identification system was used to confirm the identity of the isolates according to manufacturer’s instructions.

Antimicrobial susceptibility testing of isolates

The susceptibility of the isolates to six major antimicrobial agents was investigated using the disk diffusion method according to the Clinical and Laboratory Standards Institute 2019 guidelines.[9]

The antimicrobial agents tested were ceftazidime (30 µg), ciprofloxacin (5 µg), gentamicin (10 µg), trimethoprim-sulfamethoxazole (1.25/23.75 µg), ampicillin-sulbactam (10/10 µg) and meropenem (10 µg). Escherichia coli ATCC 25922 was used for quality control of the testing.

Phenotypic detection for extended-spectrum beta-lactamases

Any isolate from the susceptibility testing with ceftazidime zone of inhibition of ≤22 mm was assumed to be a suspected extended-spectrum beta-lactamases (ESBL) producer.[9] This was confirmed using the combination disk test screening methods as previously reported. The E-test strips of cefotaxime (16–25 µg/ml) and cefotaxime-clavulanate (1.0–0.016 µg/ml) were used. E. coli ATCC 25922 and Klebsiella pneumoniae ATCC 700603 were used for quality control of the testing.

Genotypic detection of extended-spectrum beta-lactamases

The extraction of DNA from the phenotypically ESBL-positive A. baumannii isolates was done using the alkaline lysis method as previously reported.[10] The extracted DNA was amplified using a previously published primer (TEM, SHV and CTX-M) and according to a previously optimized protocol.[11] Following completion of the polymerase chain reaction cycles, the amplicons were subjected to 2% agarose gel electrophoresis, stained with ethidium bromide and were visualized by ultraviolet transillumination.

Outbreak control measures

The hospital policy on outbreak investigation was followed which includes notification to the appropriate authorities, ward closure or cohorting of patients and staff as appropriate and institution of other infection control and prevention practices.

RESULTS

A total of eight A. baumannii isolates were identified during the study period. Six (75.0%; 6/8) of the isolates were recovered from patients, one (12.5%; 1/8) each from wash hand basin and floor of the ward. No isolate was recovered from the swabs taken either from any unit staff members or hospital items, supplies and equipment.

Figure 1 depicted the microbiologic behavior of the eight isolated pathogens in the presence of six major antimicrobial agents. It showed overall isolate susceptibility rate of 56.3% (27/48) to all the six antimicrobial agents with all (8/8; 100.00%) isolates susceptible to meropenem and 75.0% (6/8) to ampicillin-sulbactam. Conversely, the overall isolate resistance rate is 35.4% (17/48) with 62.5% (5/8) of the isolates resistant to trimethoprim-sulfamethoxazole and 50.0% (4/8) to each of ciprofloxacin and ceftazidime. Consequently, 37.5% (3/8) of the isolates were MDR as it showed resistance to the antibacterial activity of at least three different antimicrobial classes. Nonclinical isolates exhibited more AMR than their clinical counterparts (9/12%–75.0% vs. 8/36%–22.2%).

Figure 1.

Heat map of the different degrees of susceptibility of the 8 recovered Acinetobacter baumannii isolates to 6 different antimicrobial agents used in this study

Figure 2 showed the outcome of the double disk synergy test. Four (50.0%; 4/8) of the isolates were positive for ESBL production following preliminary screening but only three (37.5%; 3/8) were confirmed as phenotypically ESBL-positive. The three confirmed ESBL-producing isolates showed a greater than three two-fold (up to 8 two-fold in some cases) concentration decrease of cefotaxime (25 µg/ml) tested alone versus cefotaxime-clavulanate combination (0.23 µg/ml).

Figure 2.

Minimum inhibitory concentration of Acinetobacter baumannii with cefotaxime alone and with cefotaxime/clavulanic acid combination from Sokoto State, Nigeria

Figure 3 showed that the three ESBL-producing isolates harbored bla_SHV and bla_TEM genes and that bla_CTX-M gene was absent.

Figure 3.

Photograph of polymerase chain reaction products showing samples 1 to 3

DISCUSSION

We report the first published work both on the outbreak of A. baumannii from Sokoto, Nigeria, and detection of bla_SHV from hospitalized patients in Nigeria. Our hospital-wide (non-ICU units) surveillance revealed A. baumannii prevalence rate of 26.1% (6/23) from bla_TEM and bla_SHV resistance genes-producing clinical isolates that were 100% and 75% susceptible to meropenem and ampicillin-sulbactam, respectively.

Previous hospital-wide surveillance studies conducted in Northwestern Nigeria reported A. baumannii prevalence rates of 2.0%–6.7%.[5,12] The disparity recorded between our study and others could be attributable to differences in study designs and geographic contexts. For instance, Kalgo et al. recruited ambulatory patients from Kebbi state and reported a prevalence rate of 4.6%.[12] However, A. baumannii is a pathobiont that is more prevalent among hospitalized patients than ambulatory patients;[7,13] with those in the ICU having the highest prevalence and mortality rates.[1,8] A previous multisite retrospective study from Sokoto had reported A. baumannii prevalence rate of 2.3% among the clinical isolates of all bacterial pathogens identified. Samples (clinical but no environmental) were collected from both ambulatory and hospitalized patients in 2019 with no ESBL-producing A. baumannii strains detected.[14] Consequently, A. baumannii was likely present as a colonizer (rather than super-infection or outbreak) as its notoriety is in part due to its remarkable ESBL-producing ability.[2,7] Noteworthily, during an outbreak of A. baumannii, the pathogen can be recovered from several sites from the patient’s environment (like floor and wash hand basin of a ward) as was observed in our study.[7]

Carbapenem (like meropenem), one of the β-lactam antibiotics with the broadest spectrum and highest in-vitro activity against Gram-negative coccobacilli bacterium, exhibits concentration-independent bactericidal effect with superb safety and tolerability profiles;[7] making it the first-line agent for empirical therapy against A. baumannii infections in areas with low rates of resistant strains and last-resort antibiotics for the treatment of severe infections associated with ESBLs-producing Gram-negative organisms.[15] Little wonder that resistance to carbapenem is a marker for MDR bacteria because it correlates with a broad range of co-resistance to unrelated antibiotic classes.[3]

Meropenem is moderately more potent against Gram-negative organisms than imipenem, which is susceptible to hydrolysis by dehydropeptidase-I and may require co-administration with cilastatin.[7] We recorded 100% susceptibility rate of the MDRAB phenotypes to meropenem. However, such susceptibility rate seemed to vary according to the geographic contexts with rates of 48.9%, 29.2% and 27.1% reported nationally, continentally and globally, respectively.[3,5,16,17,18,19] Data from Meropenem Yearly Susceptibility Test Information Collection and SENTINEL programs also confirmed this geographic variation of meropenem susceptibility rates and the temporal increase in carbapenem-non susceptible clinical isolates.[20,21] The temporal decrease in carbapenem effectiveness against clinical isolates of A. baumannii has also been reported in Nigeria[5] and elsewhere.[4] Similarly, Upula et al. recorded 100% susceptibility rate to meropenem among the A. baumannii recovered from the patients with surgical site infections in Calabar, Nigeria.[22] They recruited patients from surgical wards where infection prevention and control (IPC) measures must be strictly adhered to (these wards have one of the highest risks of patients acquiring nosocomial infections due to breaks in anatomical barriers from surgical sites or catheter insertion as evinced by the six clinical isolates in our center being from patients with indwelling catheter[7]) and this might be one of the reasons both studies had similar findings. Another likely reason for the motivation to adhere to IPC is the awareness of same generated by COVID-19 pandemic.

AMR is among the top 10 global health threats adversely impacting human, animal and environmental health as well as global economy and development.[18] Consequently, AMR is an urgent, unmet medical need globally with both public health importance and socioeconomic consequences and this underscores the recent inclusion of AMR surveillance as an indicator of the UN Sustainable Development Goal Target 3.d.[3,18] We detected Ambler class A β-lactamases: bla_TEM and bla_SHV, which are two of the three major groups of ESBLs and also the most prevalent groups.[13] They are naturally resistant to penicillins, has acquired resistance to extended spectrum cephalosporins like ceftazidime and can be inhibited by beta-lactamase inhibitors like sulbactam.[15,23] bla_CTX-M, the third major group of ESBLs, was not detected among our isolates probably because it is more prevalent among community isolates,[15] although in the last decade it has become the leading ESBL in some geographic contexts.[13] While TEM-ESBLs have been harbored by the clinical isolates of A. baumannii studied in Nigeria,[6,16,24] only one published work in the country have recovered SHV-ESBLs from study participants (whom were routine antenatal attendees);[25] seemingly making TEM-ESBLs to be more prevalent than SHV-ESBLs, an amidase more likely associated with uropathogens (urine samples) and is rarely present without TEM-ESBLs. This trend was also observed abroad.[8,26,27] From the foregoing, we are the first to publish the presence of SHV-ESBLs from A. baumannii isolates from hospitalized patients in Nigeria. A. baumannii is naturally transformable to take DNA through horizontal gene transfer of resistance-determining mobile genetic elements like plasmids and this has been proposed as the likely mechanism by which sulfhydryl variable (SHV)-carrying genes were acquired from Enterobacteriaceae (examples include the ESKAPE pathogens: Enterobacter spp. and K. pneumoniae) and E. coli, which were the natural hosts of SHV-ESBLs.[15,23] Co-occurrence of multiple resistance genes in a single strain of A. baumannii is a frequent phenomenon;[26,28] and we suspect simultaneous use of the two ESBLs to be the likely reason one nonclinical isolate from our center was able to resist the action of ampicillin-sulbactam combination. This is because sulbactam is resistant to the hydrolytic actions of carbapenemases (like bla_OXA-23),[7] which are putatively more potent than when either bla_TEM or bla_SHV acts alone. Not surprisingly, the US FDA recently approved a sulbactam combination (sulbactam-durlobactam) for the treatment of MDRAB (like carbapenem-resistant A. baumannii [CRAB]).[29] As a nonclinical isolate, effective use of fimbriae and biofilm formation must be part of the strain’s survival amory,[1,5,7] thus may have overtime become more adept at its deployment relative to its clinical counterparts whom might have had shorter time. Furthermore, it is quite possible an entirely different resistance mechanism was deployed by the particular strain as evidenced by the antibacterial activity of trimethoprim-sulfamethoxazole, ceftazidime and ciprofloxacin being resisted by more than the three ESBL producers.

Even though production of beta-lactamase is reportedly the major AMR mechanism of A. baumannii, other identified mechanisms include enzymatic alteration of outer membrane proteins (like porins) and antibiotic target site and overexpression of efflux pumps, and a combination of the mechanisms could be deployed.[1]

In 2019, non-ESBL producing strains of A. baumannii were identified from the center,[14] but by 2020 an outbreak of ESBL producing strains of A. baumannii has occurred suggesting selection pressure probably due to inappropriate antimicrobial prescribing and use, and poor antimicrobial stewardship: both are among the main drivers of the emergence of AMR.[2,6,7,18] Other socioeconomic determinants of AMR (like poor water, sanitation and hygiene infrastructures, inadequate IPC practices, accumulation of antibiotics in the environment, and their use in the animal and food industries) might also be involved.

In addition, it is pertinent to point out that while other centers in Nigeria (including Northern Nigeria) are reporting the temporal increase of CRAB, our center is still having only ESBLs-producing strains of A. baumannii. This could be a testament to the high level of adherence to IPC practices and quality antimicrobial consumption (prescription and use-probably by prescribing according to the WHO AWaRe classification of antibacterial medicines) as evinced by no isolate being recovered from any of the unit personnel and the unit materials: equipment, items and supplies. Furthermore, in 2019, the A. baumannii strains were identified abroad, while in 2020, the identification was done locally and timeous suggesting improved diagnostics which contributes to improving patient outcome as no mortality was recorded during the outbreak period.

Following the report of the outbreak to the authorities and relevant stakeholders in the hospital, preventive measures such as cohorting of infected patients, health education to staff on antimicrobial stewardship, IPC practices, hand hygiene, ward cleaning, disinfection, fumigation and a temporary closure were instituted with immediate effect. Timeous changing of urinary catheters to avoid possibility of biofilm formation and lifting the catheter bags off the floor are other measures recommended to prevent re-occurrence.

Since AMR is multifactorial in nature, the One Health (“Tricycle Model”) approach might be incorporated into the measures already instituted in the study center by inaugurating an interdisciplinary committee of relevant stakeholders in the State. Consequently, the routine surveillance should include regular sample collection from cats, sewage and waste water within the hospital environment (including regular assessment of the biocides used); from student hostels and communities around the hospital environment and from veterinary clinics/hospitals, farms, event centers, amusement parks, public tap water system, and surface water within the metropolis. Furthermore, education and regular training of healthcare workers about IPC practices and AMR, sensitizing members of the community (including those affiliated to private health sector-formal and informal) about AMR, improved diagnostics using whole genome sequence or next-generation sequence and research and dissemination of findings will all help in providing evidence-informed data for tackling AMR at local, regional and national levels.

This study is limited by our inability to molecularly establish clonality of the isolates. This is due mainly to our limited molecular biology capacity. A comprehensive follow up study is however being planned in which multilocus sequence typing will be used to establish clonality of isolates from the outbreak setting.

CONCLUSION

MDR strains of A. baumannii harboring bla_SHV and bla_TEM genes were recovered from clinical and environmental sources during the period of the outbreak. The outbreak was contained with measures to prevent recurrence recommended to all relevant stakeholders.

Financial support and sponsorship

The study was self-funded by the authors.

Conflicts of interest

There are no conflicts of interest.