INTRODUCTION

Microorganisms are ubiquitous. They have been detected in several areas of the environment. Air, water, soil, and fluid from animals are carrier/vehicles of microorganisms. The quality of air is usually affected by the presence of microorganisms, which include bacteria, fungi, and viruses and people breathe in on average 14 m³ of air per day.¹ Poor air quality, especially contaminated with microorganisms, can lead to severe health challenges for humans. Microorganisms are transmitted through other routes, which include contaminated food and food products, droplet contact by sneezing, coughing or contacting with contaminated surfaces or soil. The mechanisms how microorganisms attach to animate and inanimate things have been previously reported.² Biofilms may be found on a wide variety of surfaces. Physical forces determine how microorganisms are transmitted and attached to surfaces. Once bacteria get attached to surfaces, they start to divide, resulting in biofilms, which cause the complex structure of natural sediments.³ The microorganisms may either be active and reproduce immediately or remain inactive on surfaces for long periods, making it difficult to identify the contamination source.⁴ Antibiotic resistance is a serious health challenge not only among human pathogens, but also in isolates found in other habitats. Many resistant pathogenic bacteria and commensals are found in different hosts or in the environment at large with the potential of causing infections that are usually difficult to treat.⁵ Brucella species, Shigella species, Salmonella species, Mycobacterium tuberculosis, and Neisseria meningitidis have been reported as the most common microorganisms causing laboratory-acquired infections (LAI). Infections because of the hepatitis virus, human immunodeficiency virus, and fungal infections caused by dimorphic fungi have also been commonly reported.⁶ Laboratory-acquired and nosocomial infections pose an important challenge globally and the characterization of microorganisms causing such infections is important as it provides possible therapeutic solutions for some LAI. It is important to characterize microorganisms causing LAIs to devise procedures to prevent subsequent outbreaks.⁷ This study aimed to identify isolates, revealing resistance to antibiotics in isolates from surfaces at Pharmaceutical Microbiology Laboratory, Igbinedion University Okada, and characterizing the resistance mechanisms using whole genome sequencing (WGS).

MATERIALS AND METHODS

Materials

Collection of samples

Thirty samples were obtained aseptically from the surfaces of work benches, tables, fridges, sinks, equipment, windows, and doors in pharmaceutical microbiology laboratory using sterile cotton swab sticks. One sample was obtained per surface. Sterile cotton swab sticks were soaked in sterile peptone water before sampling.

Media preparation and sterilization

Four culture media, e.g. eosine methylene blue agar, mannitol salt agar, Macconkey agar, and nutrient agar, were used in this study. All culture media were prepared and sterilized based on manufacturer’s instructions.

Isolation and identification

Ten consecutive isolates were obtained in May, 2021 from 30 sample surfaces of work benches, tables, fridges, sinks, equipment, windows, and doors in the pharmaceutical microbiology laboratory of Igbinedion University Okada in Edo State, Nigeria. Samples were aseptically collected and immediately inoculated on agar plates. Inoculated plates were incubated at 37°C for 24 h. Distinct colonies formed were randomly obtained from culture plates. Pure cultures were obtained afterwards on agar slants maintained at 4°C in the refrigerator throughout the study. Identification of isolates was carried out using standard microbiological techniques.⁸ Identities of randomly selected four isolates were subsequently confirmed by matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF-MS) (Bruker Daltonik GmbH, Bremen, Germany) analysis.

Antimicrobial susceptibility tests

The Kirby-Bauer susceptibility testing technique (Bauer et al.)⁹ was performed and results were analyzed using European Committee on Antimicrobial Susceptibility Testing (EUCAST) criteria.¹⁰ The isolates were tested using 15 antibiotics: ampicillin, meropenem, ertapenem, ceftazidime, cefotaxime, amoxicillin/clavulanic acid, cefoxitin, cefepime, cefpodoxime, tigecycline, ciprofloxacin, amikacin, piperacillin/tazobactam, cefuroxime, and gentamicin (Oxoid, Basingstoke Hampshire, UK).

Whole genome sequencing

WGS was carried out for four randomly selected isolates, whose identities were confirmed by MALDI-TOF mass spectrometry (Bruker Daltonik GmbH, Bremen, Germany) analysis. Genomic DNA (gDNA) extraction was carried out using the MagAttract HMW DNA extraction kit (Qiagen, Hilden, Germany). Quantification of gDNA was performed on a Qubit^® 2.0 fluorometer using the dsDNA BR Assay kit (Invitrogen by Thermo Fisher Scientific, Waltham, MA, USA) and diluted to 0.2 ng/µL based on the manufacturer recommendations (Illumina sample preparation guide, Illumina Inc, San Diego, CA, USA). Preparation of fragment libraries of the bacterial genomes was carried out using the Illumina Nextera XT DNA library preparation kit (Illumina Inc, San Diego, CA, USA). A DNA fragment library preparation was carried out using 1 ng of gDNA (Illumina sample preparation guide). Paired end sequencing using a read length of 2 x 300 bp on an Illumina Miseq (Miseq ver. 3.0, Illumina Inc) was performed using Miseq reagent kit v3 containing the reagent cartridge and flow cell. Pooled libraries were loaded on the reagent cartridge. Samples were sequenced to obtain a minimum average coverage of 100 fold based Illumina’s recommended standard protocols.

Raw reads (FASTQ files) were trimmed at their 5’ and 3’ ends until an average base quality of 30 was reached in a window of 20 bases, and assembly was performed using Velvet version 1.1.04 Zerbino¹¹, using optimized k-mer size and coverage cut values based on the average length of contigs with ˃1.000 bp. Species identification via MALDI-TOF MS was confirmed using ribosomal multilocus sequence typing (rMLST) (https://pubmlst.org/species-id). Identification of antimicrobial resistance genes (ARGs) was carried out using the comprehensive antibiotic resistance database-resistance gene identifier (RGI).¹² ARGs were identified on the basis of a minimum cut-off of 98% nucleotide identity for perfect or strict hits predicted by RGI. Sequences were analyzed for their plasmid replicon types using PlasmidFinder and their MLST using MLST 1.8 software, both available from the Center for Genomic Epidemiology.^13,14 No statistical analysis method was used to analyzing results.

Nucleotide sequence accession numbers

This whole genome shotgun project has been deposited at DDBJ/ENA/GenBank under the accession numbers JAJNEH000000000-JAJNEK000000000. The version described in this paper is version JAJNEH010000000-JAJNEK010000000. Table 1 shows the genome assembly statistics of the recovered isolates.

RESULTS AND DISCUSSION

Species identification using MALDI-TOF-MS and ribosomal MLST assigned 4 isolates to 4 different species (Table 2). Identified laboratory isolates include Atlantibacter hermanii, Stenotrophomonas maltophilia, Enterobacter hormaechei, and Leclercia adecarboxylata (Table 2). Out of 4 randomly selected identified isolates, 3 isolates revealed antibiotic resistance and were further analyzed by WGS (Table 3). No information about antibiotics is obtained in the EUCAST table for S. maltophilia. The laboratory isolates were ampicillin-resistant (100%), cefotaxime (67%), amoxiclav (100%), cefepime (67%), ceftazidime (67%), cefpodoxime (67%), cefuroxime (67%), ciprofloxacin (67%), gentamicin (67%), and tigecycline (67%). Two isolates were positive in the phenotypic testing of ESBLs and had ESBL gene bla_CTX-M-15 detected (Table 3).

WGS revealed that 3 out of 4 identified isolates harbored more than one resistance gene. Resistance genes were detected in S. maltophilia, but did not pass the minimum cut-off 98% nucleotide identity for perfect or strict hits predicted by RGI. The resistance determinants in the isolates included β-lactamase genes, bla_TEM-1, bla_ACT-24, bla_CTX-M-15, bla_OXA-1; aminoglycoside modifying enzymes, aac (3)-IId, aph(6)-Id, aph3”)-lb, aac(6’)-lb-cr4; qnr gene, qnrB1; sulfonamide resistance gene, sul2; tetracycline resistance gene, tet(D); phenicol resistance gene, catII and trimethoprim resistance gene, dfrA14. Other resistance determinants, which included the regulatory systems modulating antibiotic efflux CRP, antibiotic target alteration gene EF-Tu were also detected in the antibiotic resistant isolates. Table 4 shows the characteristics of the antibiotic resistant laboratory isolates. E. hormaechei isolated was of the sequence type ST 78. A. hermanii, S. maltophilia, and L. adecarboxylata had previously unknown sequence types (Table 4). Plasmids from the incompatibility group detected among the isolates were predominantly of Inc F and Col family types (Table 4).

Microbial contamination in a laboratory varies in different laboratories based on their geographical location and measures used to control infection, which poses an important challenge. Few reports have been made on this issue, especially in developing countries like Nigeria, which is really a drawback. Most of the previous studies were based on the phenotypic characterization of microbial isolates from surfaces in the laboratory. In a previous Nigerian study by Isola and Olatunji¹⁵, bacterial isolates obtained from laboratory surfaces were characterized and identified. The results showed that the most frequent microorganisms from laboratory surfaces were Bacilli. Others included Salmonella typhae and Staphylococcus aureus. Another previous study identified Staphylococcus epidermis and aerobic spore bearers, i.e. Bacillus subtilis, a common microorganism contaminating working areas in a microbiology laboratory.¹⁶ Strains identified were possible pathogens and could cause LAIs. In another study by Veena Kumari et al.¹⁷, laboratory surface samples were assessed for microbial contaminants. Out of the 60 surface samples assessed, coagulase- negative Staphylococci were the most frequent contaminant, followed by Gram-positive bacilli (Corynebacterium spp.). In our study, isolates obtained were on a relatively small scale compared to previous studies. Significantly, no Gram-positive isolate was identified among the isolates obtained in this study. All isolates were Gram-negative bacteria. More than one resistance determinant was found on the draft genome sequences of the resistant isolates, which showed that they are potential pathogens that may cause LAIs. Resistance mediated by bla_CTX-M-15 has been reported globally, including in Nigeria.^18,19,20 Recently, the first report on bla_CTX-M-15 in clinical isolates of Providencia spp., Citrobacter freundii, and Atlantibacter hermannii isolated from humans in Nigeria was published.²¹ To the best of our knowledge, we report the first genomic characterization of resistance to antibiotics in isolates obtained from surfaces of a University Science Laboratory in Nigeria. This suggests the circulation of the gene bla_CTX-M-15 in a different setting. Significantly, resistance genes were detected in L. adecarboxylata observed to be sensitive to most of the antibiotics in the susceptibility testing. This shows the importance of studying antibiotic resistance not using phenotypic methods only but further genotypic and molecular characterization techniques like WGS.

Enterobacter cloacae ST66, ST78, ST108, and ST114 strains are known to be extended spectrum cephalosporin-resistant spread internationally as high-risk clones.²² E. hormaechei ST78 isolate detected in this study belonged to the high-risk clone known particularly for the nosocomial spread carbapenemases and ESBLs.²² Jesumirhewe et al.²¹, in a recent report, detected E. hormaechei ST78 isolates in a Nigerian Hospital. E. hormaechei ST78 identified in this study suggests the circulation of this high-risk lineage in a different setting. The detection of the predominant plasmid replicon types (Inc F and Col) among the resistant isolates in this study displayed their importance in the transmission of antibiotic resistance. A recent report indicated that the IncF plasmid type is the most prevalent among human ESBL Enterobacteriaceae isolates in Nigeria.²¹

CONCLUSION

Microorganisms are ubiquitous including the laboratory environment. Techniques for reducing contamination should be employed, which include the use of soaps to clean laboratory surfaces, pre- and post- treatment of hands in disinfectant before conducting any experiment in the laboratory, the use of protective clothing, when working in the laboratory. Laboratory coats must be strictly used in the laboratory and unworn outside the laboratory. Measures should be aimed at eliminating/significantly reducing these microorganisms from laboratory using proper procedures. Frequent assessments of surfaces of the laboratory should be carried out not using phenotypic methods; only but molecular methods to identify and explore the genetic mechanisms of resistance to antibiotics in isolates, which is important to understand the dissemination of resistant isolates. Further molecular studies must characterize the prevailing clonal lineages and plasmids that harbor resistance mediating genes in the isolates. Frequent assessments would assist laboratories in either avoiding or eliminating most microbial contaminants found in the laboratory.

Ethics

Ethics Committee Approval: Not required for the study.

Informed Consent: Not required for the study.

Peer-review: Externally peer-reviewed.

Authorship Contributions

Concept: C.J., Design: C.J., Data Collection or Processing: C.J., A.O.A., Analysis or Interpretation: C.J., W.R., Literature Search: C.J., A.O.A., Writing: C.J.

Conflict of Interest: No conflict of interest was declared by the authors.

Financial Disclosure: The authors declared that this study received no financial support.