Abstract

Objective: Chryseobacterium indologenes are aerobic, Gram negative, non-fermentative rods that are intrinsically multidrug-resistant. Although an uncommon environmental pathogen, C. indologenes has been implicated recently in nosocomial infections and very resistant to plethora of antibiotics. The aim of this study is to phenotypically determine the occurrence of extended spectrum beta-lactamase (ESBL), cephalosporinase (AmpC) and metallo-beta-lactamase (MBL) production in isolates of Chryseobacterium indologenes recovered from hospital environment. Material and Methods: Hundred and eight (104) environmental samples were collected from different inanimate sources from hospital wards out of which fourteen (14) of them were from the floors and sixteen (16) of them from sinks. The isolates were identified and characterized using a commercial test kit, API 20NE^® BioMérieux, France following preliminary isolation and identification on three culture media. The identified bacteria subsequently were subjected to Antimicrobial Susceptibility Testing using Kirby Bauer agar disk diffusion method according to CLSI standard. Extended spectrum β-lactamase production was determined phenotypically by Double Disc Synergy Test (DDST) using Ceftazidime and Cefotaxime alone and in combination with Clavulanic acid. AmpC production was also determined using Cefoxitin and Ceftazidime – Double Disk Synergy Test (CC-DDST) and Ceftazidime-Imipenem antagonistic test (CIAT) methods so as to detect both inducible and constitutive AmpC production, whereas MBL detection was done by Imipenem EDTA Combined Disc Diffusion Test and using Modified Hodges Test (MHT). Results: Of the twenty isolates recovered, two (47.8%) of the bacteria isolated were C. indologenes. The two non-duplicate isolates were recovered from the floor and a sink in Paediatrics Ward and Male Surgical Ward respectively. On Mueller Hinton Agar (MHA), the bacteria produced a characteristic yellowish pigmentation. Antimicrobial susceptibility testing (AST) revealed both bacteria as multidrug-resistant to different classes of antimicrobials used against them. The organisms were resistant to Amoxiclav (100%), third generation-cephalosporins (3GC) (100%), monobactam (100%), carbapenems (100%), aminoglycosides (100%), lipopeptides (100%), and glyclycycline (100%) used. However, they were completely susceptible to piperacillin, piperacillin-tazobactam and ciprofloxacin (100%). The isolates also showed a multiple resistance index of 0.8. C. indologenes isolates recovered from hospital environment were found to phenotypically produce β-lactamases including ESBL, AmpC and MBL. Conclusion: The isolation of C. indologenes from hospital environment in this study should be a concern to us as its route into humans may be very lethal considering its multidrug-resistant nature. This study therefore encourages proper infection control measures to be advocated in health facilities, need to maintain good hygiene in hospital environment, as well as regular handwashing so as to minimize or prevent the cross transmission of this pathogen to patients as the resultant effect may be unprecedented. There may also be need for Clinical Microbiologists, Laboratory Scientists and Pathologist to routinely include the isolation and identification of these less reported non-fermenting Gram-negative bacteria in their laboratories.

1. Introduction

Chryseobacterium species are a group of non-motile, catalase-positive, oxidase-positive, indole-positive and nonglucose-fermenting Gram-negative bacilli. They produce a distinctive yellow pigment on blood agar. The genus Chryseobacterium includes six species that were previously designated as members of the genus Flavobacterium ¹. Chryseobacterium species are widely distributed in nature and in hospital environments, as well as known to live in soil, water and can be found in a wide range of Foods ^{2, 3, 4}. They have been obtained from the hospital environment, often in association with clusters of clinical isolates, as well as in municipal water sources despite effective chlorination. Chryseobacterium species are low-virulent bacteria, hence their presence in clinical samples usually indicates colonization rather than infection ⁵.

Chryseobacterium indologenes is the most commonly isolated species, however it is a rare human pathogen. This pathogen has been implicated in bacteremia caused by intravascular catheters, as well as bacteremia linked to malignancy and neutropenia ⁵. C. indologenes was formerly known as Flavobacterium indologenes, and bacteremia due to C. indologenes was first described in Taiwan ². Chryseobacterium indologenes, C. meningosepticum, and C. gleum are the most common Chryseobacterium species isolated. However, based on phylogenetic and phenotypic data, C. meningosepticum has presently been placed in the genus Elizabethkingia and is now known as E. meningoseptica ⁵.

C. indologenes can be found naturally in soil, water, plants, and food; but it is not seen in human microbiota regularly ^{3, 4, 6}. It can be found in water systems and on wet surfaces in hospitals, serving as a possible reservoir of infection. The use of indwelling devices during a hospital stay has been associated with nosocomial infections caused by C. indologenes. It has been implicated in causing major health care-associated infections such as pneumonia, empyema, pyelonephritis, cystitis, peritonitis, meningitis, and bacteremia in patients harboring central venous catheters, as well as cause life-threatening infections in individuals it has colonized through contaminated medical devices containing fluids such as respirators, endotracheal tubes, mist tents, humidifiers, infant incubators, ice chests, and syringes ⁷.

Clinically, C. indologenes has also been recovered from different patient age-groups including neonates, pre-term babies and adults, and as well has been widely reported in many continents of the world including Africa ^{7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20}. Due its low virulence, C. indologenes is known to form biofilms on foreign objects (such as indwelling devices) and exhibit protease activity, which may play a role in pathogenicity of invasive infections. It is ubiquitous and intrinsically resistant to multiple antibiotics including the carbapenems ^{8, 21, 22, 23, 24, 25, 26}.

Although some report has been made on this pathogen in the South-South and South-western part of our country ^{3, 4, 6}, no report on C. indologenes has been documented in print precisely in the South-eastern part of Nigeria to the best of our knowledge. The need to bring to light the growing presence of this organism in hospital environment and perhaps in clinical settings necessitated the importance of this study, as well as characterizing this isolate which is growing globally in its medical importance. Thus, this present study seeks to detect the presence of β-lactamase enzymes (ESBL, AmpC and MBL) in isolates of C. indologenes recovered from hospital environment of a tertiary health care facility in the South-eastern part of Nigeria.

2. Materials and Methods

This study was conducted in a tertiary health care facility in Anambra State, South-Eastern part of Nigeria within a period of one month. The tertiary health care facility serves as a referral centre to the entire State located at the centre of the State. This was part of a larger study conducted within a one-year period. A total of 104 environmental samples were collected from different sample sources/sites in our study area. All the samples used were environmental samples collected from different wards in the hospital. The samples were collected from the floor, sinks, mops, patients’ beds and tables at the Medical Ward and Surgical Ward (Male and Female) respectively as well as Paediatrics ward.

All the samples were collected maintaining aseptic condition using sterile swab sticks moistened in a sterile saline on these inanimate sources. The moistened sterile swab stick was rolled over the sample sites 3-5 seconds to collect the sample and returned aseptically into their container. These environmental samples collected were transported to the laboratory for analysis within 30 minutes of collection.

The samples were processed within an hour for identification of bacteria in the Medical Microbiology Laboratory Department, Nnamdi Azikiwe University Teaching Hospital (NAUTH) Nnewi, Anambra State. Three culture media were used for preliminary isolation of bacteria, including MacConkey Agar (MCA), Chocolate agar (CHOA) and Cetrimide agar (CA) (Titan Biotech Ltd, Rajasthan India). The media were prepared under aseptic condition following the manufacturer’s instruction. The swabs were first inoculated directly unto MCA and CHOA and Cetrimide Agar, and then sub-cultured unto Mueller-Hinton Agar (MHA) (Titan Biotech Ltd, India). Following inoculation, the media were incubated at 35°C – 37°C in air for 18 – 24 hours.

Preliminary identification was performed using standard Microbiological methods including culture, Gram stain, colonial morphology on MAC, CHOA and CA, motility test, as well as pigment production on Muller-Hinton Agar (MHA). Biochemical tests including catalase and oxidase tests were carried out on the isolates and were positive for the two tests.

Further characterization was done using API 20NE (BioMérieux, France), a commercial test kit used for identification of non-lactose fermenting bacteria. The organisms were confirmed as Chryseobacterium indologenes with 99.1% ID. E. coli ATCC 25922 and P. aeruginosa ATCC 27853 were used as control strains.

The modified Kirby-Bauer disk diffusion method was used for susceptibility testing following the guideline of Clinical Laboratory Standard Institute (CLSI) using single antibiotic discs – (drug concentrations in μg): piperacillin (100μg), cloxacillin (30μg), ceftazidime (30μg), cefotaxime (30μg), cefepime (30μg), cefoxitin (30μg), amikacin (30μg), tobramycin (10μg), netilmicin (10μg), imipenem (10μg), meropenem (10μg), aztreonam (30μg), ciprofloxacin (5μg), polymyxin B (300units), colistin (30μg), tigecycline (30μg), piperacillin/tazobactam (110μg) and ceftazidime/clavulanic acid (30μg) (Oxoid Ltd. Basingstoke, UK) on Mueller-Hinton (MH) agar plate (Titan Biotech, India). The test isolates were adjusted to 0.5 McFarland turbidity standard prior to culture and sensitivity study, and incubated at 35°C - 37°C for 18-24 h. The zone of inhibition was measured, recorded, and interpreted as susceptible (S), and resistant (R) using standard antibiotic breakpoints as stated in the CLSI ²⁷. Standard strains of Escherichia coli ATCC 25922 and P. aeruginosa ATCC 27853 were used as quality controls for the antimicrobial testing.

The MARI indexes were determined for the bacterial isolates using a previously described methodology ²⁸ according to the following formula:

The isolates were screened to determine β-lactamase resistance phenotypes present in C. indologenes. Extended spectrum-β-lactamase (ESBL) production was screened by testing the susceptibility of the isolates to any one or two of the third generation oxyimino-cephalosporins. Isolates that showed reduced susceptibility to the antimicrobial activity of Cefotaxime or Ceftazidime at inhibition zone of ≤ 23 mm were suspected to be ESBL-producer and were subjected to confirmatory tests ²⁹.

In the same vein, production of AmpC β-lactamase was inferred or suspected if any of the isolates showed reduced susceptibility to cefoxitin at inhibition zone of ≤ 18 mm and resistance to 3GC as was previously described ³⁰. Metallo-β-lactamase (MBL) producing C. indologenes isolate was suspected when the test organism(s) was resistant to any of the carbapenems including imipenem and meropenem as was described previously ³¹. Bacterial isolates showing inhibition zone diameter (IZD) of ≤ 23 mm to any of the carbapenems were suspected to produce MBL enzyme and these isolates were subjected to phenotypic confirmatory test.

The production of Extended Spectrum β-lactamase (ESβL) was confirmed by the Double Disk Synergy Test (DDST) in the isolates using Ceftazidime (30µg) and Cefotaxime (30µg) antibiotic disks with Amoxycillin-Clavulanic acid (30µg) placed at the centre 15mm apart from them. A ≥5mm increase in the inhibition zone diameter for either of the cephalosporins (ceftazidime and cefotaxime) tested in combination with amoxycillin confirms the presence of ESBL ^{29, 32}.

To confirm the presence or production of AmpC enzyme, two different methods were used including: Cefoxitin and Ceftazidime-Double Disk Synergy Test (CC-DDST) and Ceftazidime-Imipenem Antagonistic Test (CIAT) methods as was previously described ^{33, 34, 35} and with slight modifications so as to detect both constitutive and inducible AmpC production. For CC-DDST, a difference of at least 4mm in the Cefoxitin-Ceftazidime inhibition zones compared with the cefoxitin disk used alone was phenotypically confirmed the AmpC enzyme production; whereas for CIAT, antagonism was indicated by the reduction in inhibition of the zone around the Ceftazidime adjacent to the Imipenem disk or Cefoxitin disk was interpreted as being positive for AmpC producers.

Metallo-β-lactamase (MBL) production was also confirmed using two methods including Combined disc diffusion test (CDDT) using Imipenem/Imipenem+EDTA and Modified Hodges Test (MHT) using standard strain E. coli ATCC 25922 with Imipenem disc at the centre as was previously described ³¹. For the combined disc diffusion test (CDDT) using Imipenem/Imipenem+EDTA, a difference of ≥ 7 mm between the zones of inhibition of any of the carbapenems with EDTA and disks without EDTA infers MBL production phenotypically. Whereas the modified Hodges or Cloverleaf test was performed by aseptically swabbing MHA plates with Escherichia coli ATCC 25922 strain and heavily streaking the test bacteria in three different directions with imipenem disk at the centre. The plates were macroscopically observed for indentation, and the growth of the test bacteria towards the imipenem (10 µg) susceptibility disk. Growth of test bacteria towards the carbapenem disk is indicative of metallo-β-lactamase production phenotypically ^{31, 36}. Pseudomonas aeruginosa ATCC 27853 and E. coli ATCC 25378 was used as control.

All tests were performed on Mueller–Hinton agar plates already inoculated with the test C. indologenes. isolate (equivalent to 0.5 McFarland turbidity standards). All test plates were incubated at 30°C for 18-24 hours and zone of inhibition were recorded and interpreted in the CLSI criteria ²⁷. Data Analysis Statistical analysis was done using simple percentages.

3. Results

A total of sixty-five (62.5%) bacteria isolates were recovered in all out of which forty-two (64.6%) of them were non-lactose fermenters (NLF). Of the non-lactose fermenters recovered, two (47.8%) of the bacteria were confirmed as Chryseobacterium indologenes by conventional biochemical reactions and using API 20NE^® identification commercial test kit representing a prevalence of 2 (3%) of the entire bacteria recovered. These two bacteria were isolated from a sink (1/16) and floor (1/14) in the Paediatrics Surgical Ward and Male Surgical Ward respectively. On MCA, the isolates showed as non-fermenting bacteria producing yellow pinpoint colonies, while on CHOA, it produced bright yellow round colonies; however, it showed no visible growth on CA. More so, the bacteria on subculture on MHA, it produced very bright yellowish coloration on MHA, which is its major characteristic (Figure 1). A Gram’s-stained smear of the colony from CHOA and MCA revealed the bacteria as Gram-negative bacilli that were non-motile, catalase, and oxidase positive.

The antimicrobial susceptibility testing was performed and interpreted as per CLSI 2020 for Pseudomonas aeruginosa on Muller Hinton agar by Kirby Bauer disc diffusion method ²⁷. Both isolates were discovered to be susceptible to piperacillin, piperacillin-tazobactam, tigecycline and ciprofloxacin, but resistant to amoxicillin-clavulanate, ceftazidime, cefotaxime, cefepime, aztreonam, cefoxitin, amikacin, tobramycin, netilmicin, imipenem, meropenem, cloxacillin, colistin, and polymyxin (Figure 2). The resistance rates to these antibiotics were 100% and multidrug-resistant. The two C. indologenes isolates recovered were found to be positive for ESBL-production by DDST (Figure 3), positive for AmpC-production by CIAT and CC-DDST methods, and as well positive for MBL-production using both imipenem/EDTA combined double disk and MHT techniques (Figure 4).

Both C. indologenes isolates were all multi-drug resistant, and had a MARI of greater than 0.8. They were also proved to be carbapenemase producers phenotypically. The carbapenemase producers using MBL-production using both imipenem/EDTA combined double disk and MHT techniques (Figure 4).

4. Discussion

Chryseobacterium indologenes which was previously classified as Flavobacterium indologenes, a Gram-negative rod is an environmental organism. C. indologenes is a colonizer that can be found everywhere in nature (ubiquitous), mostly in soil and water ⁹. Infection by this pathogen has been reported as being rare; but it may result in significant morbidity and mortality in certain group of people. Some infections caused by this pathogen in immunocompromised patients has been reported from all over the world, and includes serious infections such as pneumonia, empyema, pyelonephritis, cystitis, peritonitis, meningitis, and bacteremia in patients harboring central venous catheters ⁷. Previous studies have also shown this organism to be multi-drug resistant to various antibiotics used against it ^{12, 14, 37, 38}.

Although paucity of data is available on the isolation of this pathogen from hospital environment, this study showed that out of sixty-five (62.5%) bacteria isolates recovered from various hospital environment sites, two isolates representing a prevalence of 3% were recovered. Previous study from Taiwan also recovered ten C. indologenes isolates from blood samples of 15 patients sampled over a 5-year period, but with a prevalence of 2.95% ³⁸. This discrepancy could be attributed to the study duration, sample nature, sampling techniques and sample size.

In this study, we isolated C. indologenes isolates were recovered from the floor and a sink (environmental sources) in two different wards in our study area. The isolation of this organism from sink and floor may not be far-fetched from the fact that they serve as reservoir of various kind of microorganisms in hospital environment; as people walk in and out of the ward continuously and use the sink to do hand washing or other kind of washing. This contrasts other previous studies which isolated this pathogen from majorly clinical samples including blood, cerebrospinal fluid, urine, sputum, endotracheal secretions bronchoalveolar brush border, rectal swab, and biliary liquid drainage of immunocompromised patients ^{10, 13, 14, 37, 38, 39}.

More so, in this study C. indologenes were recovered showed growth on MCA, CHOA and MHA, but not CA. The inability of the bacteria to grow in CA could be because it is a selective media meant primarily for isolation P. aeruginosa and contains cetrimide (cetyltrimethylammonium bromide), a selective agent that inhibits growth of other Gram-negative bacteria. In contrast most isolates of this bacteria have been achieved using Blood agar and MHA, but not MCA ^{11, 17}. Blood agar is an enriched bacterial growth medium, whereas MHA is a non-selective, non-differential medium. The use of API 20NE commercial identification kit in this study is noteworthy as this agrees with other previous studies ^{2, 16}, whereas other commercial and molecular methods such as Phoenix or Vitek 2 system, MALDI-TOF, etc. have also been used in the identification ^{12, 40, 41}.

Also, results from antibiotics susceptibility testing showed that C. indologenes isolates were completely multidrug-resistant, having shown multiple 100% resistance to amoxicillin-clavulanate, ceftazidime, cefotaxime, cefepime, aztreonam, cefoxitin, amikacin, tobramycin, netilmicin, imipenem, meropenem, cloxacillin, colistin, and polymyxin. This agrees with previous report in France which described it as an emerging multidrug-resistant bacterium ⁸ and in USA where it was reported as multidrug-resistant to aminoglycosides, cephalosporins, aminopenicillins, and aztreonam, as well as carbapenems ¹¹. They however showed susceptibility to piperacillin, piperacillin-tazobactam, tigecycline and ciprofloxacin, which collaborates previous studies ^{2, 11, 41}. The implication of this is very weighty on our health care facilities as it complicates the management course of patients who may be found to be infected with this pathogen, increases duration of hospital stay, as well as increases their cost of care.

Furthermore, the detection of β-lactamases’ (ESBL, AmpC and MBL) production in this pathogen revealed that C. indologenes phenotypically produced ESBL, AmpC, as well as MBL. This study found that this pathogen gave positive test to both CIAT and CC-DDST methods used in determining AmpC production in this study; thus, showing the capacity of this pathogen to produce AmpC β-lactamases. This could also explain the nature of multidrug-resistance found in this study. The implication of this could be that this pathogen has intrinsic ability to produce this enzyme, as well as ability to acquire this enzyme from surrounding bacteria in its ecological niche through mobile genetic elements like plasmid and integrons. This finding is supported by ¹², who in a study in Taiwan established that 80.0% of invasive C. indologenes isolates recovered were biofilm producers using the LuriaeBertani (LB) medium. This also explain why infection control in our health facilities must be scaled-up, as any means of this pathogen gaining access to patients from these hospital environment sites (such as sink, floors, etc.) either through caregivers or health workers themselves could portend great danger to the life and management of our patients.

In addition, this study revealed that C. indologenes recovered from these hospital sites were 100% resistant to the carbapenems tested against it, and as well positive for MBL-production by Imipenem/EDTA combined disk test and using MHT. The inhibition by EDTA and the ability to hydrolyse carbapenem establishes the enzyme as a metallo-β-lactamase. This is well collaborated by studies in Kuwait, Senegal, India, France, USA, and Iran ^{8, 9, 10, 11, 13, 42, 43}. Moreso, resistance to the carbapenems, which are the last line of drugs poses a big threat to our Healthcare system. This resistance could be attributed to production of the MBL (class B) gene, IND-1 by C. indologenes which has also been reported as naturally known to be resistant to β-lactam antibiotics including the carbapenems ^{19, 39, 44}.

Chryseobacterium indologenes which were successfully recovered from hospital environment in this study showed the presence of both AmpC and MBL gene production phenotypically; however, this study could not be able to characterize the presence of these genes using molecular assay.

5. Conclusion

Despite the fact that C. indologenes is a relatively rare pathogen, the number of cases documented has risen over time especially in clinical settings. The isolation and identification of this bacterium from hospital environment should serve as a wake-up call Clinical Microbiology Laboratories especially in this part of the world to include it as one of the nosocomial pathogens suspected when managing immunocompromised patients with prolonged hospital stay history and in the presentation of a defiant treatment protocol for any suspected Gram-negative infection. There is also need for infection control to evaluate the hospital environment occasionally for pathogens that could be implicated in nosocomial infections.

Ethics Approval and Consent to Participate

Ethical approval was obtained from the Research and Ethics Committee of Chukwuemeka Odumegwu Ojukwu University Teaching Hospital (COOUTH), Awka, with reference number COOUTH/CMAC/ETH.C/VOL.1/0059. All isolates used in this study were obtained from inanimate objects in the wards of COOUTH, Awka.

Statement of Competing Interests

The author(s) declare(s) that there is no conflict of interest regarding the publication of this article.

Abbreviations

CLSI: Clinical laboratory Standards Institute.

EDTA: Ethylene-diamine Tetra Acetic acid.

DDST: Double Disk Synergy Test.

CIAT: Ceftazidime-Imipenem Antagonistic Test.

CC-DDST: Cefoxitin/Ceftazidime-Double Disk Synergy Test.

CDST: Combined Disk Synergy Test.

IND-1: Indologene-1

MHT: Modified Hodges Test.

MDR: Multi-Drug Resistance.

MAC: MacConkey Agar.

CHOA: Chocolate Agar.

CA: Cetrimide Agar.

MHA: Mueller Hinton Agar

Acknowledgements

Our special appreciation goes to the laboratory scientists in Medical Microbiology and Parasitology department of NAUTH, who rendered technical assistance during the course of this research study.

References