F1000Research F1000Research 2046-1402 F1000 Research Limited London, UK 10.12688/f1000research.133298.1 Research Article Articles Phenotypic and genetic extended spectrum beta lactamase profiles of bacterial isolates from ICU in tertiary level hospital in Kenya

[version 1; peer review: 2 approved with reservations]

 Mwale Job Conceptualization Funding Acquisition Investigation Resources Writing – Original Draft Preparation Writing – Review & Editing https://orcid.org/0009-0002-4429-201X a 1 2 3 Magomere Edwin O. Data Curation Formal Analysis Validation Visualization Writing – Original Draft Preparation Writing – Review & Editing https://orcid.org/0000-0002-7735-1576 b 4 Maina Brian Investigation Methodology Software Validation https://orcid.org/0000-0002-4992-6838 5 Otieno Leon Investigation Methodology Software Validation Writing – Review & Editing 6 Onyambu Frank G. Conceptualization Data Curation Project Administration Resources Supervision Validation Writing – Review & Editing https://orcid.org/0000-0002-1195-8762 7 8 Kassim Ali Conceptualization Data Curation Project Administration Resources 3 Muchiri Lucy Conceptualization Formal Analysis Methodology Project Administration Resources Supervision Writing – Review & Editing 1 Department of Human Pathology, University of Nairobi, Nairobi, Nairobi County, Kenya Department of Laboratory Medicine, Meru Teaching and Referral Hospital, Meru, Meru County, Kenya Department of Laboratory Medicine, Kenyatta National Hospital, Nairobi, Nairobi County, Kenya Department of Biochemistry and Molecular Biology, Egerton University, Nakuru, Nakuru County, Kenya Centre of Microbiology, Washington State University, Nairobi, Nairobi county, Kenya Department of Clinical Medicine and Therapeutics, University of Nairobi, Nairobi, Nairobi County, Kenya Molecular Biosciences and Genomics Laboratory, Centre for Molecular Biosciences and Genomics, Nairobi, Nairobi County, Kenya Department of Medical Laboratory Sciences, Meru University, Meru, Meru county, Kenya mwalemagare@gmail.com emagomerre@gmail.com

No competing interests were disclosed.

 5 5 2023 2023 12 469 21 4 2023 Copyright: © 2023 Mwale J et al. 2023 This is an open access article distributed under the terms of the Creative Commons Attribution Licence, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Background: Bacterial infections in the ICUs are a threat to the lives of critically ill patients. Moreover, their vulnerable immunity predisposes them to developing bacteria-associated sepsis, further deteriorating their already fragile health. In the face of increasing drug resistance menace, the problem of bacterial infection in ICU is likely to worsen.

This study sought to assess bacterial infections in ICU setting by identifying prevalent gram-negative bacterial species and characterizing their antibiotic susceptibility patterns. Phenotypic and genetic resistance were determined among gram-negative isolates from ICU patients.

Methods: Cross-sectional samples collected from Kenyatta National Hospital ICU between January and June 2021 were cultured for subsequent analysis. Phenotypic identification of culture-positive samples was performed using VITEK 2 while Antibiotic susceptibility patterns were determined based on Antimicrobial Susceptibility Testing (AST) results. Cephalosporin-resistant gram-negative bacteria were assessed by PCR to detect the presence of ESBL genes.

Results and discussion: Out of the 168 gram-negative isolates, Acinetobacter baumanii was the most abundant (35%). Other isolates that were present at frequencies more than 15% are Klebsiella pneumoniae and Escherichia. coli. A. baumaniii is known to be a notorious bacterium in ICU due to its multidrug resistance nature. Indeed, A. baumanii isolates from Kenyatta National Hospital showed significantly high level of phenotypic resistance. Concordant with the high level of phenotypic resistance, we found high carriage of the ESBL genes among the isolates analysed in this study. Moreover, majority of isolates harboured all the four ESBL genes.

Conclusion: A high rate of phenotypic and genetic resistance was detected among the tested isolates. Resistance to cephalosporins was primarily driven by acquisition of the ESBL genes. The high-rate multidrug resistance in ICU bacterial isolates in this study has a crucial implication for ICU patient management and general antibiotics use.

 Intensive Care Unit anti-biotic susceptibility gram-negative cephalosporins Extended Spectrum Beta Lactamase Not funded Notapplicable The author(s) declared that no grants were involved in supporting this work. Background

The intensive care unit (ICU) is a hotspot of nosocomial infections primarily because of the extremely vulnerable population of critically ill patients, usage of invasive procedures such as catheters and ventilators 1 , 2 and immunosuppressive medication. 3 These infections significantly increase the burden of bacterial associated morbidity, mortality, and healthcare costs. ICU acquired infections (ICU-AI) contribute 20-25% of all nosocomial infections globally. 4 Recent studies have reported high risk of bloodstream infections caused by gram-negative bacteria, such as Escherichia coli and Klebsiella pneumoniae among COVID-19 patients admitted in ICU. 5 , 6

Antimicrobial resistance (AMR) is a major contributor to the problem of ICU acquired infections. AMR reduces the effectiveness of antibiotics and other antimicrobial drugs in treating these infections. Emergence of AMR leads to a higher risk of treatment failure, longer hospital stays, and increased mortality rates, as well as greater healthcare costs and resource utilization. 7 Drug resistant bacterial pathogens emerge and spread in the ICU environment as a result of acquisition of mutations, and selection of resistant strains, driven by indiscriminate use of antibiotics. 8 Additionally, gram-negative bacteria have evolved an intrinsic mechanism involving the production of extended spectrum beta lactamases (ESBLs) that breakdown the beta lactam antibiotics. 9 Resistance to antibiotics can be classified into: multidrug resistance (MDR), extensive drug resistance (XDR) and pan drug resistance (PDR) to reflect increasing number of antimicrobial agents affected by the resistance mechanism. 10 The outbreak and spread of COVID-19 also contributed to spread of drug resistant bacterial infections in ICU due to the increased number of patients requiring ICU admission. A high prevalence of bacterial pneumonia, 44% (n= 716) among covid 19 patients admitted in ICU has been reported. 11

Phenotypic resistance to the third generation cephalosporins (cefotaxime, ceftazidime and ceftriaxone) has been reported. 12 , 13 This resistance poses a significant public health threat since cephalosporins are valuable agents used in the management of a wide range of gram-negative infections including meningitis, Lyme disease, pseudomonas pneumonia, gram-negative sepsis, streptococcal endocarditis, melioidosis, penicillinase-producing Neisseria gonorrhoea, and gram-negative osteomyelitis. 14 Moreover, application of molecular tools to profile the ESBLs producing gram-negative bacteria have confirmed the presence of multiple ESBL genes in isolates of Klebsiella pneumonia, Escherichia coli, and Proteus species, corresponding to high-level resistance to third generation cephalosporins. 1

This study sought to profile phenotypic and genetic resistance to cephalosporin in bacteria isolated from ICU patients’ samples. Identification of bacterial species and phenotypic susceptibility patterns were conducted using VITEK 2 (bioMérieux). Phenotypically resistant isolates were confirmed by PCR genotyping.

 Methods Study design and study site

This was a cross sectional study carried out between January to June 2021 at Kenyatta National Hospital (KNH). KNH is the largest public referral and teaching hospital in Kenya with a bed capacity of approximately 1800. The hospital serves patients from the capital city with a population of over three million people. The hospital’s critical care unit department is composed of the main ICU and several other specialised units including Neurosurgery-CCU, Medical wards-CCU, Surgical ward-CCU, Neonatal-ICU, and the Casualty CCU. In this study, “ICU” to refers to both main ICU and other specialized CCUs.

 Ethical approval

This study was approved by the Kenyatta National Hospital (KNH)-University of Nairobi (UON) Ethics and research committee under the study number: P632/11/2020. Additionally, informed consent/assent were sought from participants or kin of the patient in cases of minors or unconscious patients. Written consent was obtained from next of kin for all participants but two. The two cases involved consent obtained from treating ICU physician, where the patients were incapacitated and their next of kin were unavailable to give consent. This decision was made based on the deferred consent principle backed by the following reasons

The research involves minimum harm to the participant

The deferment of consent procedure did not adversely affect the rights and welfare of the patient since the genomic testing (PCR) was carried out on the leftover bacterial isolates and not on the human DNA. These bacterial isolates are regarded as residual laboratory samples material

Patient confidentiality and data privacy was ensured by assigning unique study code to each participant. Participant metadata was collected using password protected excel data collection tool.

 Study population and sampling

Study participants included all patients admitted to various ICUs in KNH suspected to have bacterial infection during their entire period of admission. Inclusion criteria included having a gram-negative culture positive specimen. Patients with only gram-positive cultures were excluded. Sample size was determined using the Cochrane’s and Finite population correction for proportions formula. 15

AST and phenotypic detection

Sample quality and quantity were reviewed prior to labelling for bacteriology assessment. Degraded samples or those with inadequate volume were excluded. Samples that passed the inclusion criteria were processed for organism identification and antimicrobial susceptibility of culture positive gram-negative isolates using the Vitek®2 ( Biomérieux, Marcy l’Etoile, France) with Minimum Inhibitory Concentration (MIC) breakpoints set according to CLSI 2020 guidelines. Prior to loading isolates into the VITEK® 2, bacterial suspensions were prepared by emulsifying the isolates in 0.5% saline and standardizing turbidity to 0.5 McFarland’s using a densitometer. The suspension was used for species identification, AST and phenotypic detection of ESBL producing organisms in the VITEK® 2 using gram-negative cards (GN83). Vitek®2 Advanced Expert System (AES) was used. Antimicrobial susceptibility profiles for Cefotaxime, ceftazidime and ceftriaxone were also recorded. For specimens identified phenotypically as ESBL producers, another inoculum was picked from residual specimen and stored in skimmed milk-tryptone-glucose-glycerol broth at -80°C to minimize risk of mutations during batching, awaiting PCR.

 PCR Genotyping

Isolates that showed phenotypic resistance to Cefotaxime, ceftazidime and ceftriaxone were selected and used for subsequent PCR genotyping. The Isolate II Genomic DNA kit (Bioline London, UK) was used for total DNA extraction. The kit applies affinity columns to extract genomic DNA. Proteinase K, together with cell lysis buffers containing chaotropic salt ions are used to lyse cells releasing gDNA, which is captured by the affinity resins (silica gel membrane). DNA extraction was followed according to manufacturer’s instructions and eluted in a final volume of 40 ul PCR amplification was then performed using MyTaq™ PCR mix (Bioline, London, UK) in a final volume of 20μl, comprising a master-mix, 0.4 μM of each forward and reverse primers and 3 μl of DNA template. Primers specific to ESBL encoding genes (tem, shv, ctmx and oxa) were used as described by. 16 , 17

Briefly, amplicons were analysed by gel electrophoresis run in 1% agarose gel, 1×TAE buffer and SYBR™ Safe (Invitrogen, Carlsbad, CA, USA) and a 1KB ladder at 70 volts for 30 minutes. The amplified products were visualized under Ultraviolet trans-illumination using the UVTEC Gel Documentation Systems (Cleaver Scientific, United Kingdom,) to identify presence of ESBL genes. The primer sequences and thermocycling conditions used in this study are provided in the Supplementary table 1 and Supplementary table 2 in the Data Availability section (DOI: 10.6084/m9.figshare.22369975).

 Statistical analysis

Statistical analyses were performed in MS. Excel 2010 and GraphPad Prism (version 8.0.4). Shapiro-Wilk test was used to assess data normality prior to analyses. Descriptive statistics including means and frequencies were used for data summary. Mean comparisons among three or more groups was performed using one-way ANOVA with Tukey’s post-hoc. Descriptive data was presented as mean ± SD and data considered statistically significant at p value <0.05.

 Results Isolate distribution

A total of 168-gram-negative isolates were phenotypically identified from ICU patients’ samples. The isolates comprised of 8 gram-negative bacteria species, with A. baumanii being the most abundant (35%) followed by K. pneumoniae (24%), and E. coli, 18% while the remaining species were present at frequencies ≤10% ( Figure 1).

The frequency of gram-negative bacteria species identified in ICU patient samples. Phenotypic susceptibility and ESB production: Majority of isolates were ESBL producers

Phenotypic susceptibility analysis revealed high level of resistance among the bacterial isolates identified. Overall, 101/168 (60.1%) isolates were ESBL producers while 67/168 (39.9%) were ESBL non-producers. Tem was the most abundant ESBL, occurring in 99/168 followed by shv (88/168), ctmx (81/168), and oxa (54/168) ( Table 2). However, while tem was produced by most of the organisms, the differences in the differences in number of ESBL was not statistically significant ( Table 1 and Figure 2).

ESBL production by gram-negative bacterial isolates from ICU patients’ specimen.
Species tem shv ctxm oxa n
ESBL +ve ESBL -ve ESBL +ve ESBL -ve ESBL +ve ESBL -ve ESBL +ve ESBL -ve ESBL +ve ESBL -ve
C. freundii 1 2 1 2 0 3 0 3 1 2
S. marcescens 1 2 1 2 1 2 0 3 1 2
E. coloacae 3 3 3 3 3 3 2 4 3 3
K.pneum. pneumoniae 0 8 0 8 0 8 0 8 0 8
P. aeroginosa 4 13 2 15 2 15 2 15 4 13
E. coli 20 11 20 11 18 13 11 20 20 11
K. pneumoniae 32 9 32 9 31 10 19 22 32 9
A.baumanii 38 2 29 11 26 14 20 20 40 19
Total 99 50 88 61 81 68 54 95 101 67

Comparison of various types of ESBL showed that tem was the most abundant ESBL, while oxa was the least. A. baumani was the most frequent ESBL producer.

 Comparison of mean number of organisms producing various ESBLs showed no statistical significance. Bacterial abundance per specimen type: Tracheal aspirate specimen has the highest abundance of bacteria

The highest number of bacteria were isolated from tracheal aspirate (TA) (99/168) followed by urine (38/168) and blood (19/168) while ascitic tap, CVC tip and sputum had one isolates each. Species distribution analysis showed that A. baumanii were the highest in TA (38/99) Table 2.

Distribution of species per specimen type.
A. baumanii C. freundii E. coloacae E. coli K. pneumoniae K. pneumniae pneumoniae P. aeruginosa S. marcescens Total
Ascitic tap 1 0 0 0 0 0 0 0 1
Blood 9 0 3 4 3 0 0 0 19
CVC tip 0 0 0 0 0 0 1 0 1
Pus swab 0 0 0 2 2 0 3 2 9
Sputum 1 0 0 0 0 0 0 0 1
T/A 38 3 2 14 23 8 10 1 99
Urine 10 0 1 11 13 0 3 0 38
Total 59 3 6 31 41 8 17 3 168

The T/A samples had the highest number of bacterial isolates, with majority being A. baumaii isolates. T/A: Tracheal aspirate, CVC: central venous catheter.

 ESBL production and different parameters

Majority of patients 76% were males and the highest number of bacterial isolates were from patients aged between 21 to 40 years 75/168 and 50 out of the 75 isolates were phenotypically resistant to at one cephalosporin. Conversely, few isolates (3/168) were isolated from patients aged >80 years; all the isolates were phenotypically susceptible to all tested cephalosporins ( Table 3).

Summary of ESBL production and different parameters.
Positive (n=101) Negative (n=67) Total
Age ≤20 10 10 20
21 – 40 50 25 75
41 – 60 27 21 48
61 – 80 14 8 22
>80 0 3 3
Gender Male 82 46 128
Female 19 21 40
Comorbidities Positive Negative
HIV YES 2 0 2
NO 99 67 166
HYPERTENSION YES 12 1 13
NO 89 66 155
DIABETES YES 6 1 7
NO 95 66 161
COVID-19 YES 7 0 7
NO 94 67 161

Majority of patients were between 21-40 years and males were the majority. Hypertension was the most common comorbidity.

The susceptibility pattern revealed high level of phenotypic resistance against three cephalosporins (Ceftazidime, Ceftriaxone, and Cefotaxime) ( Table 4).

Susceptibility patterns of various bacterial species.
Ceftazidime Ceftriaxone Cefotaxime
S I R S I R S I R
A. baumannii 8 0 53 3 5 53 6 2 53
C. freundii 2 0 1 2 0 1 2 0 1
E. cloacae 4 0 4 3 0 5 3 0 5
E. coli 7 0 24 3 0 28 3 0 28
K. pneumoniae 7 6 34 4 0 43 4 0 43
Proteus mirabilis 0 0 1 0 0 1 0 0 1
P. aeruginosa 12 1 4 0 0 0 0 1 16
S. marcescens 2 0 1 2 0 1 2 0 1

A. baumanii had the highest resistance to all the three tested antibiotics followed by K. pneumoniae and E. coli respectively.

 Genotypic susceptibility

The 101 isolates that were phenotypically resistant to cephalosporin were subjected to PCR genotyping and 97 (96%) isolates harboured at least one of the four gene tested while only two (1.9%) isolates were negative for all the four genes. These results confirmed the phenotypic identification results by VITEK 2 ( Table 5).

Frequency of cephalosporin resistance genes per species.
A. baumanii E. cloacae E. coli K. pneumoniae P. aeruginosa
n=41 n=3 n=20 n=33 n=4
CTXM 26 (63.4) 3 (100.0) 18 (90.0) 31 (93.9) 2 (50.0)
TEM 38 (92.7) 3 (100.0) 20 (100.0) 32 (97.0) 4 (100.0)
OXA 20 (48.8) 2 (66.7) 11 (55.0) 19 (57.6) 2 (50.0)
SHV 29 (70.7) 3 (100.0) 20 (100.0) 32 (97.0) 2 (50.0)

Molecular analysis detected the ESBL genes in all the bacterial species studied.

 Discussion

Bacterial infection in the ICUs represent a major burden and safety concern for patients admitted to the ICU. 18 Patients in ICU are often critically ill and require urgent care. As a result, they are prescribed antimicrobial therapy empirically to manage their condition while waiting for culture result. 4 The World Health Organization (WHO) considers this irrational use of antimicrobial in ICU a major contributor to development of antimicrobial resistance. 19 In light of the rampant use of antibiotics in ICU, this study was conducted to evaluate the level of bacterial colonization in various sample types drawn from ICU patients and the corresponding level of antibiotic resistant gram-negative bacteria. Additionally, susceptibility to three classes of cephalosporins (Ceftazidime, Ceftriaxone and Cefotaxime) was assessed.

Acinetobacter baumanii, Klebsiela pneumoniae and E. coli were the most abundant organisms (35%, 24%, and 18% respectively). The current study corroborates previous findings that Acinetobacter species (30.9%) and Klebsiella species (29.7%) followed by Pseudomonas aeruginosa (22.9%) were the most abundant organisms in ICU environment. 20 In yet another study, Pseudomonas species was found to be high (29.1%) in ICU setting followed by Acinetobacter (27.5%). 21 The trend in ICU bacterial colonization appears to be dominated by the three main organisms Acinetobacter species, Klebsiella species and Pseudomonas species, as demonstrated in previous studies 20 , 21 and corroborated by our study. Unsurprisingly, we reported Acinetobacter baumanii and Klebsiela pneumoniae as the most common organisms in ICU and resistant to all tested cephalosporins. The resistance to multiple cephalosporins might partially explain the high abundance of these bacteria in ICU. Our findings were in agreement with Saxena and colleagues’ findings that Acinetobacter and Klebsiella were resistant to multiple antibiotics. 4

Organism distribution varied significantly among different specimen types. Tracheal aspirate had the highest isolates (59%) followed by urine (23%) and blood (11%) while ascitic tap, CVC tip and sputum had (0.6%) each. These findings agreed with high prevalence (56%) of pulmonary colonization among ICU patients identified by tracheal aspirate culture. 22 Tracheal aspirate culture has been evaluated as a non-invasive method for diagnosis of ventilator-associated pneumonia colonization. 23 The ease of obtaining tracheal aspirate sample and availability of established protocol could explain why more tracheal aspirate samples were obtained and cultured successfully. Urine, blood and pus swabs yielded 23%, 11% and 5% of total organisms respectively. The lower proportion of culture positivity could be influenced by the small number of samples as well as the culture method.

Concordant with phenotypic susceptibility findings, we reported high level of genetic resistance in A. baumanii, K. pneumoniae and E. coli. A. baumanii is an opportunistic nosocomial pathogen that presents resistance to most antimicrobial. 24 This ability makes it the most persistent bacteria in ICU and has been linked to ventilator-associated pneumonia. 25 Carbapenem resistance in A. baumanii is mediated by class D β-lactamases belonging to OXA-type. Moreover, A. baumanii possesses an intrinsic chromosomally encoded oxacillinase blaOXA- 51, which may account for the high prevalence of blaOXA (48.8%) reflecting its ability to resist eradication. 26 We report 57.6% of K. pneumoniae isolates that possess OXA gene. Similar findings were recently reported demonstrating the involvement of blaOXA gene in mediating resistance to cephalosporins. 27

Analysis of bla CTMX, bla TEM, and bla SHV genes revealed a high resistance gene carriage in more than 50% of studied isolates. High prevalence of these markers had been reported previously in an Indonesian hospital. 28 Moreover, molecular surveillance of ESBL in neonates drawn from Kenya and Nigeria revealed a high prevalence of ESBL producing bacteria. 29 The high prevalence of ESBL producing bacteria in ICU underscore the need to heighten antibiotic resistance surveillance to provide the much-needed information to combat resistance to antibiotics.

The problem of increasing antimicrobial resistance in ICU is worrisome particularly due to the fragile nature of this category of patients. While there are no definitive measures to eradicate antibiotic resistant micro-organisms in ICUs, vaccines against these pathogens remain elusive and where available, they are unaffordable. Thus, prudent use of antibiotics in ICU to avoid widespread resistance is recommended. This study highlights the abundance of cephalosporin resistant gram-negative bacteria in ICU, which emphasized the need to heighten the fight against antibiotic resistance.

 Data availability

Figshare. Phenotypic and genetic Extended Spectrum Beta Lactamase cephalosporin resistance profiles of bacterial isolates from ICU in Tertiary Level Hospital in Kenya. DOI: https://doi.org/10.6084/m9.figshare.22369975.v2 (Mwale, 2023).

This project contains the following data:

Extended Spectrum Beta Lactamase.xlsx: The data contain phenotypic antibiotic susceptibility values for bacterial isolates and genotypic resistance data assessed by detection of ESBL genes is also part of the data.

Raw DATA_VITEK Bacterial identification.xlsx: Bacterial identification readings from VITEK 2.

Supplementary Materials.docx: This file contains the PCR primer sequences and thermocycler conditions.

Data are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0).

 Acknowledgement

The authors thank the study participants and their families.

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Antimicrob. Resist. Infect. Control. 2021;10:22. 33514432 10.1186/s13756-020-00871-x PMC7844809 Giannitsioti E Louka C Mamali V : Bloodstream infections in a COVID-19 Non-ICU department: Microbial epidemiology, resistance profiles and comparative analysis of risk factors and patients’ outcome. Microorganisms. 2022;10:1314. 35889033 10.3390/microorganisms10071314 PMC9318208 Tran GM Ho-Le TP Ha DT : Patterns of antimicrobial resistance in intensive care unit patients: a study in Vietnam. BMC Infect. Dis. 2017;17:429. 28619105 10.1186/s12879-017-2529-z PMC5472965 Reygaert WC : An overview of the antimicrobial resistance mechanisms of bacteria. AIMS microbiology. 2018;4:482–501. 31294229 10.3934/microbiol.2018.3.482 PMC6604941 Ferreira CM Ferreira WA Almeida NCOS : Extended-spectrum beta-lactamase-producing bacteria isolated from hematologic patients in Manaus, State of Amazonas, Brazil. Braz. J. Microbiol. 2011;42:1076–1084. 24031725 10.1590/S1517-83822011000300028 PMC3768795 Cantón R Gijón D Ruiz-Garbajosa P : Antimicrobial resistance in ICUs: an update in the light of the COVID-19 pandemic. Curr. Opin. Crit. Care. 2020;26:433–441. 10.1097/MCC.0000000000000755 Conway M Andrew Kohler K : Co-infection and ICU-acquired infection in COVID-19 ICU patients: a secondary analysis of the UNITE-COVID data set. Crit. Care. 2022;26:236. 35922860 10.1186/s13054-022-04108-8 PMC9347163 Lester R Musicha P Van Ginneken N : Prevalence and outcome of bloodstream infections due to third-generation cephalosporin-resistant Enterobacteriaceae in sub-Saharan Africa: a systematic review. J. Antimicrob. Chemother. 2020;75:492–507. 31742611 10.1093/jac/dkz464 PMC7021093 Nagassar RP Daniel K Bridgelal-Nagassar RJ : Surveillance of phenotypic extended spectrum beta-lactamase resistance in blood isolates at a Hospital in East Trinidad. Caribb. Med. J. 2021. 10.48107/CMJ2021.04.008 Arumugham VB Gujarathi R Cascella M : Third generation cephalosporins. 2019. Israel GD : Determining sample size. 1992. Kiiru J Kariuki S Goddeeris BM : Analysis of β-lactamase phenotypes and carriage of selected β-lactamase genes among Escherichia coli strains obtained from Kenyan patients during an 18-year period. BMC Microbiol. 2012;12:111–155. 10.1186/1471-2180-12-155 Saisi H Makobe C Kangongo M : Prevalence of CTXM, SHV, TEM AND OXA Genes among Extended-Spectrum Beta-Lactamase Producing Klebsiella pneumoniae from Mukuru Slum, Kenya. Adv. Microbiol. 2019;09:853–862. 10.4236/aim.2019.910052 Patil RK Kabera B Muia CK : Hospital acquired infections in a private paediatric hospital in Kenya: a retrospective cross-sectional study. Pan Afr. Med. J. 2022;41. WHO: Regional strategy on prevention and containment of antimicrobial resistance, 2010. 2010. 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Microbiol. 2014;52:3597–3604. 25078907 10.1128/JCM.01494-14 PMC4187781 Sanz-García F Gil-Gil T Laborda P : Coming from the wild: multidrug resistant opportunistic pathogens presenting a primary, not human-linked, environmental habitat. Int. J. Mol. Sci. 2021;22:8080. 34360847 10.3390/ijms22158080 PMC8347278 Kramer A Schwebke I Kampf G : How long do nosocomial pathogens persist on inanimate surfaces? A systematic review. BMC Infect. Dis. 2006;6:1–8. 10.1186/1471-2334-6-130 Roca I Espinal P Vila-Farrés X : The Acinetobacter baumannii oxymoron: commensal hospital dweller turned pan-drug-resistant menace. Front. Microbiol. 2012;3:148. 10.3389/fmicb.2012.00148 Al-Sheboul SA Al-Moghrabi SZ Shboul Y : Molecular characterization of carbapenem-resistant Acinetobacter baumannii isolated from intensive care unit patients in Jordanian hospitals. Antibiotics. 2022;11:835. 35884089 10.3390/antibiotics11070835 PMC9311868 Candra IK Yanto F Suranadi IW : Characteristic of Extended Spectrum β-Lactamase-Producing Enterobacteriaceae from Fecal Carriage Isolates of Intensive Care Unit Patients at Sanglah Hospital, Bali, Indonesia. The Open Microbiology Journal. 2021;15:1–6. 10.2174/1874285802115010001 Edwards T Williams CT Olwala M : Molecular surveillance reveals widespread colonisation by carbapenemase and extended spectrum beta-lactamase producing organisms in neonatal units in Kenya and Nigeria. medRxiv, 2022.2001.2006.22268735. 2022. 10.1101/2022.01.06.22268735 10.5256/f1000research.146277.r316326 Reviewer response for version 1 GANIYU Tajudeen Oladunni 1 Referee https://orcid.org/0000-0003-3706-2398 Fountain University, Osogbo, Osun, Nigeria

Competing interests: No competing interests were disclosed.

 16 10 2024 Copyright: © 2024 GANIYU TO 2024 This is an open access peer review report distributed under the terms of the Creative Commons Attribution Licence, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. recommendation approve-with-reservations

The research concept is okay and relevant to the scope of the journal but the manuscript needs major revision. My comments are listed below:

The authors should rewrite the abstract to include a clear statement of problem and justification. The conclusion is not emphatic on what needed to be done to address the problem.

ESBL are more than four, what are the reasons for limiting the number of gene assessed to just four? State the four genes correctly in the abstract.

In the background, third paragraph and third sentence- presence of multiple ESBL genes in isolates of Klebsiella pneumoniae, Escherichia coli and Proteus species- please list the ESBL genes.

In the Results

 - how did you differentiate ESBL producers from non ESBL producers?

- tem should be written as TEM, shv should be SHV, ctmx should be CTX-M and oxa should be OXA.

5. In the second paragraph of the results (Phenotypic susceptibility and ESB production-  the last sentence, - the differences in the differences in number of ESBL- delete in word in red.

6. Discussion.

- the discussion is poor, despite explaining and interpreting the results, the authors failed to compare their results with those of other authors. Comparing their work with those of other authors will bring out the novelty in their work and also show whether their results are better compared to earlier works.

OTHERS

E. coloacae should be Enterobacter cloacae

REFERENCES

References earlier than 2019 should be updated.

Is the work clearly and accurately presented and does it cite the current literature?

Partly

If applicable, is the statistical analysis and its interpretation appropriate?

Yes

Are all the source data underlying the results available to ensure full reproducibility?

Yes

Is the study design appropriate and is the work technically sound?

Yes

Are the conclusions drawn adequately supported by the results?

Partly

Are sufficient details of methods and analysis provided to allow replication by others?

Partly

Reviewer Expertise:

Medical Microbiology and Microbial Biotechnology

I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard, however I have significant reservations, as outlined above.

Magomere Edwin Egerton University, Kenya

Competing interests: There are no competing interests to disclose

 10 11 2024

Comment 1: The authors should rewrite the abstract to include a clear statement of problem and justification. The conclusion is not emphatic on what needs to be done to address the problem. 

Response: We have rewritten the abstract to include the problem statement in the abstract, which now reads as follows: 

Problem statement : Bacterial infections in the Intensive Care Units are a threat to the lives of critically ill patients. Their vulnerable immunity predisposes them to developing bacteria-associated sepsis, deteriorating their already fragile health. In the face of increasing antibiotics resistance, the problem of bacterial infection in ICU is worsening.

Justification: Surveillance of bacterial infections in ICUs and drug resistance will help to understand the magnitude of the problem it poses and inform response strategies.

 

Comment 2: ESBL are more than four, what are the reasons for limiting the number of gene assessed to just four? State the four genes correctly in the abstract.

Response: These four ESBL genes were chosen for PCR genotyping since they were most frequently detected based on phenotypic resistance detection. Also, we restricted our study to 4 genes due to financial constraints

Comment 3: In the background, third paragraph and third sentence- presence of multiple ESBL genes in isolates of  Klebsiella pneumoniae, Escherichia coli and  Proteus species- please list the ESBL genes.

Response: ​We have added the various ESBL genes as recommended and included a reference to it.

Comment 4: How did you differentiate ESBL producers from non ESBL producers?

Response: We used the Phenotypic detection of extended-spectrum β-lactamase production. Specifically, we used the automated VITEK 2 ESBL test ( Biomérieux, Marcy l’Etoile, France).

Description of how the method work

This method is based on the simultaneous assessment of the antibacterial activity of cefepime, cefotaxime and ceftazidime, measured either alone or in the presence of clavulanate. This test relies on card wells containing 1.0 mg/L of cefepime, or 0.5 mg/L of cefotaxime or ceftazidime, either alone or associated with 10 or 4 mg/L of clavulanate, respectively. After inoculation, cards are introduced into the VITEK 2 machine, and for each antibiotic tested, turbidity is measured at regular intervals. The proportional reduction of growth in wells containing a cephalosporin combined with clavulanate is then compared with that achieved by the cephalosporin alone and is interpreted as ESBL-positive or – negative through a computerized expert system. 

 We have also edited the subtitle on phenotypic detection to " Antimicrobial Susceptibility Testing (AST) and phenotypic detection of ESBL producers".

Comment 5: tem should be written as TEM, shv should be SHV, ctmx should be CTX-M and oxa should be OXA.

Response: We have edited the names of ESBL gene in all instances where they were not written in the right format. Thank you for drawing my attention to this.

Comment 6: In the second paragraph of the results (Phenotypic susceptibility and ESB production-  the last sentence, - the differences in the differences in number of ESBL- delete in word in red.

Response: We have deleted the words in red and re-written the sentence to read “ bla TEM was produced by most of the organisms but there were no statistically significant difference when compared to other ESBLs ”

Comments: Discussion.

- the discussion is poor, despite explaining and interpreting the results, the authors failed to compare their results with those of other authors. Comparing their work with those of other authors will bring out the novelty in their work and also show whether their results are better compared to earlier works.

Response: We have edited the discussion to discuss our findings in context of other works in the same area and added more references. Throughout the discussion, we have endeavored to compare results to previous studies.

 10.5256/f1000research.146277.r217591 Reviewer response for version 1 Kibwana Upendo O 1 Referee Muhimbili University of Health and Allied Sciences, Dar es Salaam, Tanzania

Competing interests: No competing interests were disclosed.

 6 6 2024 Copyright: © 2024 Kibwana UO 2024 This is an open access peer review report distributed under the terms of the Creative Commons Attribution Licence, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. recommendation approve-with-reservations

The work is relevant but needs major revision to be more understandable and reproducible

See comments below:

1. The authors have to be mindful of how to write scientific names properly (italicized), how to write gram stain " Gram" instead of gram, and correct all the typos. Also how the gens are written, in capital form not small letters.

2. In the abstract the authors are talking about four genes, please indicate clearly which genes are referred to.

3. The authors focus on ESBL genes. is OXA and ESBL gene or beta lactam gene? Please clarify and possibly change the title accordingly.

4. In the abstract the authors are talking about high level of multidrug resistance. Can the authors clarify what  MDR is? because this is not indicated anywhere in the manuscript nor is it reflected in the results.

5. It is not clear if this was a laboratory-based cross-sectional study or simply cross cross-sectional study. At some point, it seems the starting point was in the lab with Gram-negative isolates, while in the population section, the authors indicate they started in the ward (ICU) please indicate clearly. If the study started in the lab what was the rationale for obtaining informed consent rather than requesting a waiver for informed consent? If the study started from the ward how did you exclude pats with Gram-positive isolates prior to sample collection?

6. The authors have to explain how they obtained isolates which subjected to identification test on VITEK. Please indicate all quality control measures observed.

7.How many antibiotics were tested during AST? Provide the names and conc.

8. The results section has to be rearranged to provide a better narrative. Start with where the isolates were obtained from, which isolates were isolated from where, then narrow down to the isolated ESBL.

9. Most of the results sections can be improved to describe better what is presented in the tables. e.g. the bacterial abundance section can be improved by adding information on the isolates which were found in one type of specimen only. The ESBL   production and different production parameters section can be improved by adding information  about  other parameters; Would be good to perform a chi-square test to see if the differences between the parameters are significant or coincidental due to numbers.

10. genotypic susceptibility section has to be described more; how many isolates carried one gene only? How many multiple? Which was the most combo of genes? In which isolates were more prominent with multiple genes etc. Also why 2 isolates were negative and not 4? 97 were positive.

11. "The susceptibility pattern revealed high level of phenotypic resistance against three cephalosporins (Ceftazidime, Ceftriaxone, and Cefotaxime)" This sentence seems ectopic where it is placed.

12. In the discussion section the authors should mention the settings of the studies that they make comparisons with e.g. in paragraph two.

13. "Unsurprisingly, we reported Acinetobacter baumanii and Klebsiela pneumoniae as the most common organisms in ICU and resistant to all tested cephalosporins" This statement appears in the discussion section yet it is not indicated anywhere in your result section. Please discuss the things that are from your results.

14. Table 1 'n' should be 'total'.

15. Table 2 should be table 1. This is based on a comment about rearrangement.

16. "Urine, blood and pus swabs yielded 23%, 11% and 5% of total organisms respectively. The lower proportion of culture positivity could be influenced by the small number of samples as well as the culture method." Please explain the about the culture methods referred to in this statement.

17. Can the authors compare their genotypic results with neighbor countries and give clinical significance.

18. I advice the authors to remove the footnotes on the tables because they are rather captions and not footnotes

19. Table 4 is not clear. What are the numbers provided? Percentages or numbers? Provide numbers and percentages. Table not clear? What about other abx? What is the number of tested isolates for each species?

20. The title for table 5 is not clear. What information does the table present?

Is the work clearly and accurately presented and does it cite the current literature?

Partly

If applicable, is the statistical analysis and its interpretation appropriate?

Partly

Are all the source data underlying the results available to ensure full reproducibility?

Yes

Is the study design appropriate and is the work technically sound?

Yes

Are the conclusions drawn adequately supported by the results?

Partly

Are sufficient details of methods and analysis provided to allow replication by others?

Partly

Reviewer Expertise:

Bacteriology, infectious diseases

I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard, however I have significant reservations, as outlined above.

Magomere Edwin Egerton University, Kenya

Competing interests: No competing interests to declear

 11 11 2024

Reviewer 1 comments

Comment 1: The authors have to be mindful of how to write scientific names properly (italicized), how to write gram stain " Gram" instead of gram, and correct all the typos. Also how the genes are written, in capital form not small letters.

Response: All the typos have been corrected throughout the manuscript  and gene names written in the right format

Typos: “gram stain” corrected to “Gram stain”,

Italicized all scientific names

Specific gene names have been added to the abstract: bla TEM, bla CTX-M, bla SHV and bla OXA.

Comment 2: In the abstract the authors are talking about four genes, please indicate clearly which genes are referred to.

Response: the genes we are referring to include: bla TEM, bla CTX-M, bla SHV and bla OXA. These have been added to the abstract.

Comment 3: The authors focus on ESBL genes. is OXA and ESBL gene or beta lactam gene? Please clarify and possibly change the title accordingly.

Response: OXA is an ESBL gene. Other ESBL genes that we studied include: ( bla CTX-M, bla SHV, bla TEM). We focused on four ESBL genes.

Response: 4. In the abstract the authors are talking about high level of multidrug resistance. Can the authors clarify what  MDR is? because this is not indicated anywhere in the manuscript nor is it reflected in the results.

Response: We have added the definition of MDR: Lines in the background section. The last sentence of conclusion under abstract has been rephrased to read as: “The high prevalence rate of ESBL genes  in ICU bacterial isolates shown in this study has a important implication for ICU patient management and general antibiotics use”.

Comment 5: It is not clear if this was a laboratory-based cross-sectional study or simply cross cross-sectional study. At some point, it seems the starting point was in the lab with Gram-negative isolates, while in the population section, the authors indicate they started in the ward (ICU) please indicate clearly. If the study started in the lab what was the rationale for obtaining informed consent rather than requesting a waiver for informed consent? If the study started from the ward how did you exclude pats with Gram-positive isolates prior to sample collection?

Response: The starting point of the study was the laboratory. However, we used patient samples that had been collected from ICU patients for clinical purposes. Since the samples were not originally collected for research purposes, KNH-UON Ethical review committee tasked us to obtain consent before samples could be used for research purposes. We also needed patient consent to allow us collect patient information such as co-morbidities.

We used differential media to allow only Gram-negative bacteria to grow. Thus, we used only Gram- negative cultures for our downstream experiments.

Comment 6: The authors have to explain how they obtained isolates which subjected to identification test on VITEK. Please indicate all quality control measures observed.

Response: Samples brought to the laboratory requested by the ICU clinical team were cultured and subjected to Gram stain. Only Gram-negative isolates were analyzed on VITEK. We used commercially acquired Gram negative isolates as positive controls for each run on Vitek. For negative controls we used bacterial suspension media (saline).

Comment 7: How many antibiotics were tested during AST? Provide the names and conc.

Response: Antibiotics tested in this study include Cefotaxime, ceftazidime and ceftriaxone. The list of tested antibiotics has been added to the manuscript. The Minimum Inhibitory Concentrations (MICs) were set according to CLSI 2020 guidelines.

Comment 8: The results section has to be rearranged to provide a better narrative. Start with where the isolates were obtained from, which isolates were isolated from where, then narrow down to the isolated ESBL.

Response: The results have been rearranged to start with various sources of isolates as advised

Comment 9: Most of the results sections can be improved to describe better what is presented in the tables. e.g. the bacterial abundance section can be improved by adding information on the isolates which were found in one type of specimen only. The ESBL   production and different production parameters section can be improved by adding information about other parameters; Would be good to perform a chi-square test to see if the differences between the parameters are significant or coincidental due to numbers.

Response 1: we have added more information about species identified in specimen types with the highest number of species including the following:

T/A harboured all the isolated tested, with a total of 99 isolates. The distribution of species in T/A showed that A. baumanii were the highest in TA (38/99). Urine specimen had the second highest number of species (38/168). Out of the 8 species identified, urine had 5 species, with K.  pneumoniae being the most frequent, identified 13 times.

Response 2: Table 3 has been replaced to show the outcome of Chi-Square analysis to show differences in various parameters

Comment 10: genotypic susceptibility section has to be described more; how many isolates carried one gene only? How many multiple? Which was the most combo of genes? In which isolates were more prominent with multiple genes etc. Also why 2 isolates were negative and not 4? 97 were positive.

Response: Added information on

. bla TEM was the most predominant gene at 96% (97/101), followed by SHV = 85.6% (86/101), CTX-M = 78.8% (80/101) and OXA= 52.9% (54/101). bla TEM / bla SHV / bla CTX-M / bla OXA and bla TEM / bla SHV / bla CTX-M gene combinations were present at 49% (n=49/101) and 25.7% (n=26/101) respectively. Other common gene combinations included blaTEM/SHV at 6.7% (n=7/101), blaTEM/CTX-M/OXA at 1.9% (n=2/101), blaTEM/SHV/OXA at 1.9% (n= 2/101), blaSHV/CTX-M at 1.9% (n= 2/101).

Comment 11:  "The susceptibility pattern revealed high level of phenotypic resistance against three cephalosporins (Ceftazidime, Ceftriaxone, and Cefotaxime)" This sentence seems ectopic where it is placed.

Response: This sentence has been deleted as advised

Comment 12: In the discussion section the authors should mention the settings of the studies that they make comparisons with e.g. in paragraph two.

Response: The settings of previous studies have now been mentioned in the discussion section

Comment 13: "Unsurprisingly, we reported  Acinetobacter baumanii and  Klebsiela pneumoniae as the most common organisms in ICU and resistant to all tested cephalosporins" This statement appears in the discussion section yet it is not indicated anywhere in your result section. Please discuss the things that are from your results.

Response: This sentence has been deleted

Comment 14: Table 1 'n' should be 'total'.

Response: “n” has been edited to “Total”

Comment 15: Table 2 should be table 1. This is based on a comment about rearrangement.

Response: Tables have been rearranged accordingly

Comment 16: "Urine, blood and pus swabs yielded 23%, 11% and 5% of total organisms respectively. The lower proportion of culture positivity could be influenced by the small number of samples as well as the culture method." Please explain the about the culture methods referred to in this statement.

Comment 17: Can the authors compare their genotypic results with neighbor countries and give clinical significance.

Response: Results have been compared to those obtained from neighboring countries (Tanzania and Uganda) and Clinical significance highlighted .

Comment 18: I advice the authors to remove the footnotes on the tables because they are rather captions and not footnotes

Response: Footnotes have been removed from all tables as advised

Comment 19: Table 4 is not clear. What are the numbers provided? Percentages or numbers? Provide numbers and percentages. Table not clear? What about other abx? What is the number of tested isolates for each species?

Response: Number provided in the table is number of isolates in the categories of S-susceptible, I-intermediate and R-resistant. This has been clarified in the revised manuscript.

Comment 20: The title for table 5 is not clear. What information does the table present?

Response: The title has been edited to read as “The isolates that had resistance to all tested cephalosporins and the frequency of resistance genes”.